Controlled dissolution crosslinked prote in crystals

ABSTRACT

The present invention relates to crosslinked protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment of said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in oxidation-reduction potential of the solution, change in the incident radiation, change in transition metal concentration, change in flouride concentration, change in free radical concentration, change in metal chelater concentration, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate. This invention also provides methods for producing such crosslinked protein crystals, methods using them for protein delivery and methods using them in cleaning agents, including detergents, pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to crosslinked protein crystalscharacterized by the ability to change from insoluble and stable form tosoluble and active form upon a change in the environment surroundingsaid crystals, said change being selected from the group consisting ofchange in temperature, change in pH, change in chemical composition,change from concentrate to dilute form, change in oxidation-reductionpotential of the solution, change in the incident radiation, change intransition metal concentration, change in flouride concentration, changein free radical concentration, change in metal chelater concentration,change in shear force acting upon the crystals and combinations thereof.According to one embodiment of this invention, such crosslinked proteincrystals are capable of releasing their protein activity at a controlledrate. This invention also provides methods for producing suchcrosslinked protein crystals, methods using them for protein delivery,methods for using them in cleaning agents, including detergents,therapeutic formulations, pharmaceutical compositions, vaccines,personal care compositions, including cosmetics, veterinarycompositions, foods, feeds, diagnostics and formulations fordecontamination.

BACKGROUND OF THE INVENTION

[0002] Proteins are used in a wide range of applications in the fieldsof industrial chemistry, pharmaceuticals, veterinary products, cosmeticsand other consumer products, foods, feeds, diagnostics anddecontamination. At times, such uses have been limited by constraintsinherent in proteins themselves or imposed by the environment or mediain which they are used. Such constraints may result in poor stability ofthe proteins, variability of performance or high cost. In order forproteins to realize their full potential in the fields in which they areused, they must be able to function without excessive intervention bytheir surrounding environment. In the past, environmental elements haveoften posed barriers to the widespread use of proteins.

[0003] Various approaches have been employed to overcome these barriers.However, these approaches have incurred either loss of protein activityor the additional expense of protein stabilizing carriers orformulations.

[0004] One unique approach to overcoming barriers to the widespread useof proteins is crosslinked enzyme crystal (“CLEC™) technology [N. L. St.Clair and M. A. Navia, J. Am. Chem. Soc., 114, pp. 4314-16 (1992)].Crosslinked enzyme crystals retain their activity in environments thatare normally incompatible with enzyme function. Such environmentsinclude prolonged exposure to proteases and other protein digestionagents, high temperature or extreme pH. In such environments,crosslinked enzyme crystals remain insoluble and stable.

[0005] Protein solubility, leading to controlled release or dissolutionof protein, is important in many industrial and medical fields. Suchfields include those concerning cleaning agents, including detergents,pharmaceuticals, including therapeutics and vaccines, consumer andpersonal care products, veterinary products, foods, feeds, diagnosticsand decontamination. Various approaches to controlled release have beenproposed. These include encapsulation, such as that described in U.S.Pat. Nos. 4,579,779 and 5,500,223. Other approaches include the use ofmechanical or electrical feed devices and osmotic pumps.

[0006] Controlled release in the pharmaceutical field has been addressedby various means. U.S. Pat. No. 5,569,467 refers to the use of sustainedrelease microparticles comprising a biocompatible polymer and apharmaceutical agent, which is released as the polymer degrades. U.S.Pat. No. 5,603,956 refers to solid, slow release pharmaceutical dosageunits comprising crosslinked amylase, alpha amylase and a pharmaceuticalagent. U.S. Pat. No. 4,606,909 refers to oral, controlled-releasemultiple unit formulations in which homogeneous cores containingparticles of sparingly soluble active ingredients are coated with apH-sensitive erodable coating. U.S. Pat. No. 5,593,697 refers topharmaceutical or veterinary implants comprising a biologically activematerial, an excipient comprising at least one water soluble materialand at least one water insoluble material and a polymer film coatingadapted to rupture at a predetermined period of time after implant.

[0007] The objective of controlled release of proteins, however, must bebalanced with the fact that the protein itself may not be stable understorage conditions. Protein stability may also be adversely affected byother components of the formulation in which it is contained. Forexample, heavy duty liquid detergents constitute hostile environmentsfor component enzymes. Such problems have been approached through theuse of mutant subtilisin proteases, which are said to have improvedoxidative stability. See U.S. Pat. No. 4,760,025 and PCT patentapplication WO89/06279. Proteins, the enzymes most widely used indetergents, catalyze their own decomposition. Strategies such as theaddition of protease inhibitors (e.g., borate with glycols) or thelowering of water activity have been only partially effective.

[0008] Another approach, described in U.S. Pat. No. 5,385,959, isencapsulation of degradation-sensitive detergent components in capsulesof composite emulsion polymers, which permit dilution release thereof.U.S. Pat. No. 5,286,404 refers to a liquid detergent composition said tohave improved enzyme solubility while preserving enzyme activity. Theimprovement is attributed to chemical modification of free primary aminogroups in an enzyme solution via aldehyde treatment, acylation oralkylation.

[0009] Despite such progress in protein technology generally, the needstill exists for proteins which are stable under conditions of storage,while active under conditions of use.

DISCLOSURE OF THE INVENTION

[0010] The present invention relates to crosslinked protein crystalscharacterized by the ability to change from an insoluble and stable formto a soluble and active form upon a change in the environmentsurrounding said crystals, said change being selected from the groupconsisting of change in temperature, change in pH, change in chemicalcomposition, change from concentrate to dilute form, change inoxidation-reduction potential of the solution, change in the incidentradiation, change in transition metal concentration, change in flourideconcentration, change in free radical concentration, change in metalchelater concentration, change in shear force acting upon the crystalsand combinations thereof. According to one embodiment of this invention,such crosslinked protein crystals are capable of releasing their proteinactivity at a controlled rate.

[0011] Advantageously, crosslinked protein crystals according to thisinvention are insoluble and stable under storage conditions and solubleand active under conditions of use.

[0012] This invention also provides cleaning agents, includingdetergents, therapeutic proteins pharmaceutical compositions, vaccines,personal care compositions, including cosmetics, veterinarycompositions, foods, feeds, diagnostics and formulations fordecontamination. Additionally, this invention includes methods forproducing such crosslinked protein crystals and methods for proteindelivery using them.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph representing the stability of various enzymes inCiba detergent #16 at 40° C.

[0014]FIG. 2 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, on fabric soiledwith blood, milk and carbon black.

[0015]FIG. 3 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at30° C., on fabric soiled with cocoa.

[0016]FIG. 4 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at40° C., on fabric soiled with cocoa.

[0017]FIG. 5 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at30° C., on fabric soiled with blood, milk and carbon black.

[0018]FIG. 6 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at40° C., on fabric soiled with blood, milk and carbon black.

[0019]FIG. 7 is a graph representing the washing performance of liquiddetergent formulations, including a formulation containing crosslinkedsubtilisin crystals according to the present invention, after storage at30° C., on fabric soiled with blood.

[0020]FIG. 8 is a graph representing the solubility of crosslinkedsubtilisin crystals according to the present invention at 30° C.

[0021]FIG. 9 is a graph representing the solubility of crosslinkedsubtilisin crystals according to the present invention at 37° C.

DETAILED DESCRIPTION OF THE INVENTION

[0022] In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following terms or phrases are employed:

[0023] Aqueous-organic solvent mixture—a mixture comprising n % organicsolvent, where n is between 1 and 99 and m % aqueous, where m is 100-n.

[0024] Biphasic substrate—A solution of a substrate with two distinctphases, either liquid/solid or liquid/liquid phases, one of whichcontains a substrate for a reaction catalyzed by the protein constituentof a crosslinked protein crystal. An emulsion of olive oil in an aqueoussolution containing discrete aqueous and organic phases exemplifies asubstrate for crosslinked crystals of lipase.

[0025] Catalytically effective amount—an amount of crosslinked proteincrystals of this invention which is effective to treat, protect, repairor detoxify the area to which they are applied over some period of time.

[0026] Chance in chemical composition—any change in the chemicalcomponents of the environment surrounding the crosslinked proteincrystals that affects the environment or the crosslinker, includingaddition of chemical reagents, chemical changes induced by applicationof energy in the form of light, microwave, or radiation to theenvironment, chemical events that affect the crosslinker andcombinations thereof.

[0027] Change in shear force acting upon the crystals—any change infactors of the environment surrounding the crosslinked protein crystalsunder conditions of use, such as, changes in mechanical pressure, bothpositive and negative, revolution stirring, centrifugation, tumbling,mechanical agitation and filtration pumping.

[0028] Controlled dissolution—dissolution of crosslinked proteincrystals or release of the protein constituent from the crystallinestate to the soluble state that is (1) triggered by a change in theenvironment surrounding said crystals, said change being selected fromthe group consisting of change in temperature, change in pH, change inchemical composition, change from concentrate to dilute form, change inoxidation-reduction potential of the solution, change in the incidentradiation, change in transition metal concentration, change in flourideconcentration, change in free radical concentration, change in metalchelater concentration, change in shear force acting upon the crystalsand combinations thereof and (2) controlled by a factor selected fromthe group consisting of the following: degree of crosslinking of saidcrosslinked protein crystals, the amino acids residues involved in thecrosslinks, whether the crosslinker is homobifunctional orheterobifunctional, the length of time of exposure of protein crystalsto the crosslinking agent, the rate of addition of crosslinking agent tosaid protein crystals, the nature of the crosslinker, the chain lengthof the crosslinker, the surface area of said crosslinked proteincrystals, the size of said crosslinked protein crystals, the shape ofsaid crosslinked protein crystals and combinations thereof. As usedherein, the phrase “controlled dissolution” does not include leaching.

[0029] Controlled dissolution crosslinked protein crystals—crosslinkedprotein crystals that slowly dissolve after being exposed a giventrigger and release the soluble form of the protein into solution. Theactivity of controlled dissolution crosslinked protein crystals arisesprimarily from the soluble form of the protein released from thecrystal.

[0030] Crosslinked crystal form of protein—crosslinked protein crystalsthat remain insoluble and in the solid state when added to solution.

[0031] Enhanced Protein activity—activity of the crosslinked crystalform of a protein which is enhanced as compared with the soluble form ofthe protein. According to various embodiments of the present invention,enhanced protein activity is exhibited by any one of the following: acrosslinked crystal form of a protein having activity that is 200-300times higher than that of the soluble form of the protein; a crosslinkedcrystal form of a protein having activity that is 100-200 times higherthan that of the soluble form of the protein; a crosslinked crystal formof a protein having activity that is 10-100 times higher than that ofthe soluble form of the protein; a crosslinked crystal form of a proteinhaving activity that is 20-50 times higher than that of the soluble formof the protein; a crosslinked crystal form of a protein having activitythat is 10-20 times higher than that of the soluble form of the protein;a crosslinked crystal form of a protein having activity that is 20-30times higher than that of the soluble form of the protein; a crosslinkedcrystal form of a protein having activity that is 5-10 times higher thanthat of the soluble form of the protein; a crosslinked crystal form of aprotein having activity that is 2-3 times higher than that of thesoluble form of the protein; a crosslinked crystal form of a proteinhaving activity that is at least 3 times higher than that of the solubleform of the protein; a crosslinked crystal form of a protein havingactivity that is at least 2 times higher than that of the soluble formof the protein; a crosslinked crystal form of a protein having activitythat is at least 25-99% higher than that of the soluble form of theprotein; a crosslinked crystal form of a protein having activity that isat least 25-30% higher than that of the soluble form of the protein; ora crosslinked crystal form of a protein having activity that is at least20% higher than that of the soluble form of the protein.

[0032] Formulations for decontamination—formulations selected from thegroup consisting of: formulations for decontamination of chemicalwastes, herbicides, insecticides, pesticides, environmental hazards andchemical warfare agents.

[0033] Insoluble and stable form of a protein—a form of a protein whichis insoluble in aqueous solvents, organic solvents or aqueous-organicsolvent mixtures and which displays greater stability than the solubleform of the protein. According to an alternate embodiment of thisinvention, the phrase “insoluble and stable form of a protein” may be aprotein which is insoluble in dry formulations but soluble in wetformulations. In any embodiment, the crosslinked protein crystals may beactive in insoluble form. And in one embodiment, the crosslinked proteincrystals may be active in insoluble form, then dissolve or are removedor digested once their function is complete.

[0034] Macromolecular substrate—a large biomolecule, such as a proteinor a carbohydrate having a molecular weight of at least 600-700 Daltons,which is also a substrate for a reaction catalyzed by the proteinconstituent of a crosslinked protein crystal.

[0035] Organic solvents—any solvent of non-aqueous origin.

[0036] Pharmaceutically effective amount—an amount of crosslinkedprotein crystals which is effective to treat a condition in anindividual to whom they are administered over some period of time.

[0037] Prophylactically effective amount—an amount of crosslinkedprotein crystals which is effective to prevent a condition in anindividual to whom they are administered over some period of time.

[0038] Protein—a protein or, alternatively, a glycoprotein or,alternatively, any peptide having a tertiary structure.

[0039] The protein constituents of the crosslinked protein crystalformulations of this invention may be naturally or syntheticallymodified. They may be glycoproteins, phosphoproteins, sulphoproteins,iodoproteins, methylated proteins, unmodified proteins or contain othermodifications.

[0040] The protein constituent of the crosslinked protein crystalformulations of this invention may be any protein including, forexample, hormones, such as parathyroid hormone, enzymes, antibodies,viral receptors, viral surface glycoproteins, parasite glycoproteins,parasite receptors, T-cell receptors, MHC molecules, immune modifiers,tumor antigens, mucins, inhibitors, growth factors, trophic factors,cytokines, lymphokines, cytokines, toxoids, nerve growth hormones, bloodclotting factors, adhesion molecules, multidrug resistance proteins,adenylate cyclases, bone morphogenic proteins and lectins.

[0041] Also included among proteins are glycoprotein hormones andcytokines. Examples of hormones include follicle stimulating hormone,human chorionic gonadotropin, luteinizing hormone, thyrotrophin andovine, bovine, porcine, murine and rat alleles of these hormones.Examples of cytokine glycoproteins include a-interferon, lymphotoxin,and interleukin-2. Also included are glycoprotein tumor-associatedantigens, for example, carcinoembryonic antigen (CEA), human mucins,her-2/neu, and prostate-specific antigen (PSA) [R. A. Henderson and O.J. Finn, Advances in Immunology, 62, pp. 217-56 (1996)].

[0042] Protein activity—an activity selected from the group consistingof binding, catalysis, or activities which generate a functionalresponse within the environment in which the protein is used, such asthe induction of an immune response and immunogenicity or hydrolysis oflipids in lipase deficient individuals, or combinations thereof.

[0043] Protein activity release rate—the quantity of protein dissolvedper unit time.

[0044] Soluble form of Drotein—individual protein molecules in solutionand dissociated from a crystal lattice.

[0045] Small molecule substrate—molecules having molecular weightsgenerally less than 600 Daltons which are also substrates for reactionscatalyzed by the protein constituents of crosslinked protein crystals.

[0046] Therapeutic protein—A protein which is administered to a patientin a pharmaceutical formulation and manner. Therapeutic proteinsinclude, for example, hormones, enzymes including lipase, antibodies,viral receptors, T-cell receptors, chemokines, chemokine receptors, MHCmolecules, tumor antigens, mucins, inhibitors, growth factors, trophicfactors, cytokines, lymphokines, toxoids, nerve growth hormones, bloodclotting factors, adhesion molecules, multidrug resistance proteins,adenylate cyclases and bone morphogenic proteins.

[0047] Vaccine antigen—a protein derived from an infectious agent suchas a virus, parasite, or tumor antigen. The protein activity of suchvaccine antigens is to induce protective immunity against the infectiousagent.

[0048] The crosslinked protein crystals of this invention areparticularly advantageous because they are stable in harsh environmentsimposed by the formulations or compositions in which they are employedor conditions of their storage. At the same time, these crosslinkedprotein crystals are capable of controlled dissolution or release oftheir activity when exposed to one or more triggers in theirenvironment. Such triggers may be selected from the group consisting ofchange in temperature, change in pH, change in chemical composition,change from concentrate to dilute form, change in shear force actingupon the crystals and combinations thereof. Controlled dissolution orrelease of activity of crosslinked protein crystals according to thisinvention may also be triggered over a change in time.

[0049] Specific examples of such triggers include an increase ordecrease in temperature, for example, an increase in temperature from alow temperature between about 0° C. and about 20° C. to a hightemperature between about 25° C. and about 70° C. Other triggers includea change from acidic pH to basic pH and a change from basic pH to acidicpH. Examples of triggers of change from concentrate to dilute forminclude, for example, a change in solute concentration, a change inconcentration of all solutes from about 2-fold to about 10,000-fold, achange in concentration of all solutes from about 2-fold to about700-fold, an increase or decrease in salt concentration, an increase ordecrease in water concentration, an increase or decrease in organicsolvent concentration, a decrease in protein concentration and adecrease in detergent concentration.

[0050] Additional triggers involve changes in chemical composition ofthe environment surrounding the crosslinked protein crystals that affectthe environment or the crosslinker itself. Such changes include, forexample, addition of chemical reagents, increase or decrease in organicsolvent concentration, chemical events that affect the crosslinker,chemical changes induced by application of energy, including light,microwave or radiation. As explained above, any of these triggers mayact in combination or in sequence with one or more of the othertriggers.

[0051] Controlled dissolution of crosslinked protein crystals accordingto the present invention may also be effected by a change in timesufficient to permit a protein activity release rate between about 0.1%per day and about 100% per day, a change in time sufficient to permit aprotein activity release rate between about 0.01% per hour and about100% per hour and a change in time sufficient to permit a proteinactivity release rate between about 1% per minute and about 50% perminute.

[0052] Crosslinked protein crystals according to this invention,therefore, include those capable of releasing their protein activity ata controlled rate upon exposure to a change in their environment, saidchange being selected from the group consisting of change in pH, changein solute concentration, change in temperature, change in chemicalcomposition, change in shear force acting upon the crystals andcombinations thereof. Said controlled rate of releasing protein activitymay be determined by a factor selected from the group consisting of thefollowing: degree of crosslinking of the crosslinked protein crystals,length of time of exposure of protein crystals to the crosslinkingagent, the rate of addition of crosslinking agent to the proteincrystals, the nature of the crosslinker, the chain length of thecrosslinker, the amino acids residues involved in the crosslinks,whether the crosslinker is homobifunctional or heterobifunctional, thesurface area of the crosslinked protein crystals, the size of thecrosslinked protein crystals, the shape of the crosslinked proteincrystals and combinations thereof.

[0053] As a result of their crystalline nature, the crosslinked proteincrystals of this invention achieve uniformity across the entirecrosslinked crystal volume. This uniformity is maintained by theintermolecular contacts and chemical crosslinks between the proteinmolecules constituting the crystal lattice. The protein moleculesmaintain a uniform distance from each other, forming well-defined stablepores within the crosslinked protein crystals that facilitate access ofsubstrate to the protein, as well as removal of product. In thesecrosslinked protein crystals, the lattice interactions, when fixed bychemical crosslinks, are particularly important in providing stabilityand preventing denaturation, especially in storage, under conditionsincluding harsh environments created by components of compositions inwhich the crystals are used. At the same time, the protein crystals arecrosslinked in such a way that they dissolve or release their proteinactivity upon exposure to a trigger in their environment encounteredunder conditions of use. Thus, they may be substantially insoluble andstable in a composition under storage conditions and substantiallysoluble and active under conditions of use of said composition.

[0054] Factors contributing to the release rate of protein activity ofcrosslinked protein crystals according to this invention include thedegree of crosslinking of the crosslinked protein crystals, the lengthof time of exposure of protein crystals to the crosslinking agent, therate of addition of crosslinking agent to the protein crystals, thelength of time of exposure of protein crystals to the crosslinkingagent, the nature of the crosslinker, the amino acids residues involvedin the crosslinks, whether the crosslinker is homobifunctional orheterobifunctional, the chain length of the crosslinker, the surfacearea of the crosslinked protein crystals, the size of the crosslinkedprotein crystals, the shape of the crosslinked protein crystals andcombinations thereof.

[0055] In addition to their activity, crosslinked protein crystalsaccording to this invention are particularly stable and insoluble understorage conditions, including the attendant storage temperature, storagepH, storage time, storage concentrate form, storage involving little orno shear force acting upon the crystals, or combinations thereof.Advantageously, these crosslinked protein crystals are soluble andactive under conditions of use, including conditions involving change intemperature, change in pH, change in chemical composition, change fromconcentrate to dilute form, change in shear force acting upon thecrystals and combinations thereof. Such properties make the crosslinkedprotein crystals of this invention particularly useful for delivery ofcleaning agents, including detergents, therapeutic proteins,pharmaceuticals, personal care agents or compositions, includingcosmetics, vaccines, veterinary compositions, foods, feeds, diagnosticsand formulations for decontamination.

[0056] According to one embodiment, the crosslinked protein crystals ofthis invention are characterized by a half-life of activity understorage conditions which is greater than at least 2 times that of thesoluble form of the protein that is crystallized to form the crystalsthat are crosslinked and activity similar to that of the soluble form ofthe protein under conditions of use.

[0057] According to one embodiment of this invention, crosslinkedprotein crystals are characterized by activity which is similar to thatof their soluble or uncrosslinked crystallized counterparts underconditions of use. Advantageously however, the crosslinked proteincrystals of this invention display improved stability under storageconditions, as compared to their soluble or uncrosslinked crystallizedcounterpart proteins.

[0058] One advantage of controlled dissolution crosslinked proteincrystals is that a trigger is required to release the soluble form ofthe protein from the crystal lattice. Therefore, controlled dissolutioncrosslinked protein crystals can be prepared and function as crosslinkedprotein crystals with protein activity in the solid crystalline stateuntil a trigger is encountered. After the trigger is encountered,soluble protein is released and protein activity derives from both thecrosslinked crystal form, as well as the soluble form of the protein.

[0059] According to one embodiment of this invention, advantageousproperties are obtained from crosslinked protein crystals that areprepared as controlled dissolution crosslinked protein crystals, butwhich are not subsequently exposed to a trigger. In particular, in theabsence of an appropriate trigger to initiate dissolution, suchcrosslinked protein crystals may exhibit enhanced protein activity tomacromolecular substrates, biphasic substrates or small moleculesubstrates as compared with their soluble counterparts. For example,lipase crystals crosslinked withsulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate(Sulfo-LC-SMPT), which are not subsequently exposed to a trigger tobreak the —S—S— bonds of the crosslinks, exhibit enhanced hydrolysisactivity toward a biphasic olive oil substrate.

[0060] More particularly, lipase crystals crosslinked withsulfo-LC-SMPT, are characterized by about a five to ten fold higherprotein activity for hydrolysis of an olive oil sustrate than thesoluble form of the protein that is crystallized to form the crystalsthat are crosslinked. In addition, lipase crystals crosslinked with1-ethyl-3-[3-dimethylaminoproplyl]carbodiimide hydrochloride (EDC) arecharacterized by about almost two fold higher protein activity forhydrolysis of an olive oil sustrate than the soluble form of the proteinthat is crystallized to form the crystals that are crosslinked.

[0061] The protein constituent of the crosslinked protein crystals ofthis invention may be any protein, including, for example, therapeuticproteins, prophylactic proteins, including antibodies, cleaning agentproteins, including detergent proteins, personal care proteins,including cosmetic proteins, veterinary proteins, food proteins, feedproteins, diagnostic proteins and decontamination proteins. Includedamong such proteins are enzymes, such as, for example, hydrolases,isomerases, lyases, ligases, transferases and oxidoreductases. Examplesof hydrolases include thermolysin, elastase, esterase, lipase,nitrilase, amylase, pectinase, subtilinase, hydantoinase, asparaginase,urease, subtilisin and other proteases and lysozyme. Examples of lyasesinclude aldolases and hydroxynitrile lyase. Examples of oxidoreductasesinclude peroxidase, laccase, glucose oxidase, alcohol dehydrogenase andother dehydrogenases. Other enzymes which may be crystallized andcrosslinked include cellulases and oxidases.

[0062] Examples of therapeutic or prophylactic proteins includehormones, enzymes, including lipase, antibodies, inhibitors, growthfactors, trophic factors, cytokines, lymphokines, toxoids,erythropoietin, Factor VIII, insulin, glucagon like peptide-I(insulinotropin), amylin, TPA, dornase-α, α-1-antitripsin, human growthhormones, nerve growth hormones, parathyroid hormone, bone morphogenicproteins, urease, toxoids, fertility hormones, FSH, LSH, postridicalhormones, tetanus toxoid, diptheria toxoid.

[0063] The crosslinked protein crystals of this invention may be used inany of a number of chemical processes. Such processes include industrialand research-scale processes, such as organic synthesis of specialtychemicals and pharmaceuticals. Enzymatic conversion processes includeoxidations, reductions, additions, including esterifications andtransesterifications, hydrolyses, eliminations, rearrangements, andasymmetric conversions, including stereoselective, stereospecific andregioselective reactions.

[0064] Thus, crosslinked protein crystals according to this inventionmay be advantageously used instead of conventional soluble orimmobilized proteins in cleaning agents, including detergents,pharmaceuticals, therapeutics, veterinary compounds, personal carecompositions, including cosmetics, foods, feeds, vaccines, pulp, paperand textile processing, diagnostics and formulations fordecontamination.

[0065] Crosslinked protein crystals according to this invention may alsobe used in various environmental applications. They may be used in placeof conventional soluble or immobilized proteins for environmentalpurposes, such wide area decontamination of environmental hazards.

[0066] Alternatively, the crosslinked protein crystals of this inventionmay be used in cleaning agents, selected from the group consisting ofdetergents, such as powdered detergents and liquid detergents, bleaches,household cleaners, hard surface cleaners, industrial cleaners andcarpet and upholstery shampoos.

[0067] Cleaning agents containing crosslinked protein crystals accordingto the present invention may also comprise compounds conventionallyincluded in such agents. See, for example, Soaps and Detergents, ATheoretical and Practical Review, Louis Spitz (Ed.), AOCS Press(Champlain, Ill.) (1996). Such compounds include anionic, non-ionic,cationic or zwitterionic surfactants, or mixtures thereof.

[0068] Anionic surfactants are exemplified by alkyl sulfates, alkylether sulfates, alkyl sulfonates, alkylaryl sulfonates, olefinsulfonates, alkyl ether phosphates, alkyl ether phosphates, fatty acidsalts, soaps, isothionates and sulfonated unsaturated esters and acids.

[0069] Non-ionic surfactants are exemplified by products of condensationof an organic aliphatic or alkyl aromatic hydrophobic compound with analkylene oxide, alkyl polyglucosides and sugar esters.

[0070] Cationic surfactants are exemplified by quarternary ammoniumsalts of tertiary alkyl amines, amino amides, amino esters orimidazolines containing al least one long chain (C₈-C₂₂) aliphatic groupor an alkyl-aryl group, wherein alkyl comprises about 4 to 12 carbonatoms and aryl is preferably a phenylene group.

[0071] Zwitterionic surfactants are exemplified by derivatives ofquarternary ammonium, quarternary phosphonium or tertiary sulfoniumcompounds, derivatives of secondary and tertiary amines and derivativesof heterocyclic secondary and tertiary amines.

[0072] And crosslinked protein crystals according to this invention maybe used as ingredients in personal care compositions, includingcosmetics, such as creams, lotions, emulsions, foams, washes, compacts,gels, mousses, slurries, powders, sprays, pastes, ointments, salves,balms, drops, shampoos, and sunscreens. In topical creams and lotions,for example, they may be used as humectants or for skin protection,softening, bleaching, cleaning, deproteinization, lipid removal,moisturizing, decoloration, coloration or detoxification. They may alsobe used as anti-oxidants in cosmetics.

[0073] According to this invention, any individual, including humans andother mammals, may be treated in a pharmaceutically acceptable mannerwith a pharmaceutically effective or a catalytically effective amount ofcrosslinked protein crystals for a period of time sufficient to treat acondition in the individual to whom they are administered over someperiod of time. Alternatively, individuals may receive aprophylactically effective or a catalytically effective amount ofcrosslinked protein crystals of this invention which is effective toprevent a condition in the individual to whom they are administered oversome period of time.

[0074] Such crosslinked protein crystals may be administered alone, aspart of a pharmaceutical, personal care or veterinary preparation or aspart of a prophylactic preparation, such as a vaccine, with or withoutadjuvant. They may be administered by parenteral or non-parenteralroute. For example, they may be administered by oral, pulmonary, nasal,aural, anal, dermal, ocular, intravenous, intramuscular, intraarterial,intraperitoneal, mucosal, sublingual, subcutaneous, or intracranialroute. In either pharmaceutical, personal care or veterinaryapplications, crosslinked protein crystals may be topically administeredto any epithelial surface. Such epithelial surfaces include oral,ocular, aural, anal and nasal surfaces, to treat, protect, repair ordetoxify the area to which they are applied.

[0075] The present invention also includes controlled releaseformulations comprising crosslinked protein crystals according to thisinvention. In such formulations, the crosslinked protein crystals aresubstantially insoluble under storage conditions and capable ofreleasing their protein activity in vivo at a controlled rate. Forexample, a pharmaceutical controlled release formulation according tothis invention, administered by oral route, is characterized in that thecomponent crosslinked protein crystals are substantially insoluble undergastric pH conditions and substantially soluble under small intestine pHconditions. Alternatively, for these and other uses according to thisinvention, the crosslinked protein crystals may be active in theinsoluble form and then dissolve and are removed or digested once theirfunction is complete.

[0076] Pharmaceutical, personal care, veterinary or prophylacticcompositions comprising crosslinked protein crystals according to thisinvention may also be selected from the group consisting of tablets,liposomes, granules, spheres, microparticles, microspheres and capsules.

[0077] For such uses, as well as other uses according to this invention,crosslinked protein crystals may be formulated into tablets. Suchtablets constitute a liquid-free, dust-free form of crosslinked proteincrystal storage which are easily handled and retain acceptable levels ofactivity.

[0078] Alternatively, the crosslinked protein crystals may be in avariety of conventional depot forms employed for administration toprovide reactive compositions. These include, for example, solid,semi-solid and liquid dosage forms, such as liquid solutions orsuspensions, gels, creams, balms, emulsions, lotions, slurries, powders,sprays, foams, pastes, ointments, salves, balms and drops.

[0079] Compositions or formulations comprising the crosslinked proteincrystals of this invention may also comprise any conventional carrier oradjuvant used in pharmaceuticals, personal care compositions orveterinary formulations. These carriers and adjuvants include, forexample, Freund's adjuvant, ion exchangers, alumina, aluminum stearate,lecithin, buffer substances, such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium, trisilicate, cellulose-based substances andpolyethylene glycol. Adjuvants for topical or gel base forms mayinclude, for example, sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol andwood wax alcohols.

[0080] According to one embodiment of this invention, crosslinkedprotein crystals may be combined with any conventional materials usedfor controlled release administration. Such materials include, forexample, coatings, shells and films, such as enteric coatings andpolymer coatings and films.

[0081] The most effective mode of administration and dosage regimen offormulations or compositions comprising crosslinked protein crystals ofthis invention will depend on the effect desired, previous therapy, ifany, the individual's health status or status of the condition itselfand response to the crosslinked protein crystals and the judgment of thetreating physician or clinician. The crosslinked protein crystals may beadministered in any dosage form acceptable for pharmaceuticals, personalcare compositions or veterinary formulations, at one time or over aseries of treatments.

[0082] The amount of the crosslinked protein crystals that-may becombined with carrier materials to produce a single dosage form willvary depending upon the particular mode of administration, formulation,dose level or dose frequency. A typical preparation will contain betweenabout 0.01% and about 99%, preferably between about 1% and about 50%,crosslinked protein crystals (w/w).

[0083] Upon improvement of the individual's condition, a maintenancedose of crosslinked protein crystals may be administered, if necessary.Subsequently, the dosage or frequency of administration, or both, may bereduced as a function of the symptoms, to a level at which the improvedcondition is retained. When the condition has been alleviated to thedesired level, treatment should cease. Individuals may, however, requireintermittent treatment on a long-term basis upon any recurrence of thecondition or symptoms thereof.

[0084] An alternate embodiment of the present invention includes proteindelivery systems comprising the crosslinked protein crystals disclosedherein. Such a system may be used to deliver proteins such as thoseincluded in cleaning agents, such as detergents, personal care products,such as cosmetics, pharmaceuticals, such as lipase, veterinarycompositions, vaccines, foods, feeds, diagnostics and formulations fordecontamination. Protein delivery systems of this invention, which maybe formulations or devices, such as implantable devices, may bemicroparticulate protein delivery systems, wherein the crosslinkedprotein crystals have a longest dimension between about 0.01 μm andabout 500 μm, alternatively between about 0.1 μm and about 50 μm. Thecrosslinked protein crystal components of such systems may have a shapeselected from the group consisting of: spheres, needles, rods, plates,such as hexagons and squares, rhomboids, cubes, bipryamids and prisms.Advantageously, the crosslinked crystal form of the proteins of thisinvention allow loading of up to between about 50% and about 90% proteinper unit of weight.

[0085] One example of a protected protein system according to thisinvention is suitable for storage in a medium such as a liquiddetergent, prior to use. The crosslinked protein crystal components ofsuch a system are insoluble under storage conditions in saidmedium—which typically causes degradation of the soluble form of theprotein that is crystallized to form said crystal that iscrosslinked—and soluble under conditions of use.

[0086] According to the present invention, preparation of crosslinkedprotein crystals includes the steps of crystallizing and crosslinkingthe protein. This may be carried out as illustrated below.

[0087] Preparation of Crosslinked Protein

[0088] Crystals—Protein Crystallization

[0089] Protein crystals are grown by controlled crystallization ofprotein out of aqueous solution or aqueous solution-containing organicsolvents. Conditions to be controlled include, for example, the rate ofevaporation of solvent, the presence of appropriate co-solutes andbuffers, pH and temperature. A comprehensive review of the variousfactors affecting the crystallization of proteins has been published byMcPherson, Methods Enzymol., 114, pp. 112-20 (1985).

[0090] McPherson and Gilliland, J. Crystal Growth, 90, pp. 51-59 (1988)have compiled comprehensive lists of proteins and nucleic acids thathave been crystallized, as well as the conditions under which they werecrystallized. A compendium of crystals and crystallization recipes, aswell as a repository of coordinates of solved protein and nucleic acidstructures, is maintained by the Protein Data Bank at the BrookhavenNational Laboratory [http//www. pdb.bnl.gov; Bernstein et al., J. Mol.Biol., 112, pp. 535-42 (1977)]. These references can be used todetermine the conditions necessary for crystallization of a protein, asa prelude to the formation of an appropriate crosslinked proteincrystal, and can guide the crystallization strategy for other proteins.Alternatively, an intelligent trial and error search strategy can, inmost instances, produce suitable crystallization conditions for manyproteins, provided that an acceptable level of purity can be achievedfor them [see, e.g., C. W. Carter, Jr. and C. W. Carter, J. Biol. Chem.,254, pp. 12219-23 (1979)].

[0091] For use in crosslinked protein crystals according to thisinvention, the large single crystals which are needed for X-raydiffraction analysis are not required. Microcrystalline showers aresuitable.

[0092] For example, the crosslinked protein crystals may have a longestdimension between about 0.01 μm and about 500 μm, alternatively, between0.1 μm and about 50 μm. They may also have a shape selected from thegroup consisting of: spheres, needles, rods, plates, such as hexagonsand squares, rhomboids, cubes, bipryamids and prisms.

[0093] In general, crystals are produced by combining the protein to becrystallized with an appropriate aqueous solvent or aqueous solventcontaining appropriate crystallization agents, such as salts or organicsolvents. The solvent is combined with the protein and may be subjectedto agitation at a temperature determined experimentally to beappropriate for the induction of crystallization and acceptable for themaintenance of protein activity and stability. The solvent canoptionally include co-solutes, such as divalent cations, co-factors orchaotropes, as well as buffer species to control pH. The need forco-solutes and their concentrations are determined experimentally tofacilitate crystallization.

[0094] In an industrial-scale process, the controlled precipitationleading to crystallization can best be carried out by the simplecombination of protein, precipitant, co-solutes and, optionally, buffersin a batch process. As another option, proteins may be crystallized byusing protein precipitates as the starting material. In this case,protein precipitates are added to a crystallization solution andincubated until crystals form. Alternative laboratory crystallizationmethods, such as dialysis or vapor diffusion, can also be adopted.McPherson, supra and Gilliland, supra, include a comprehensive list ofsuitable conditions in their reviews of the crystallization literature.

[0095] Occasionally, incompatibility between the crosslinking agent andthe crystallization medium might require exchanging the crystals into amore suitable solvent system.

[0096] Many of the proteins for which crystallization conditions havealready been described, may be used to prepare crosslinked proteincrystals according to this invention. It should be noted, however, thatthe conditions reported in most of the above-cited references have beenoptimized to yield, in most instances, a few large, diffraction qualitycrystals. Accordingly, it will be appreciated by those of skill in theart that some degree of adjustment of these conditions to provide a highyielding process for the large scale production of the smaller crystalsused in making crosslinked protein crystals may be necessary.

[0097] Preparation of Crosslinked Protein

[0098] Crystals—Crosslinking of Protein Crystals

[0099] Once protein crystals have been grown in a suitable medium theycan be crosslinked. Crosslinking results in stabilization of the crystallattice by introducing covalent links between the constituent proteinmolecules of the crystal. This makes possible transfer of the proteininto an alternate environment that might otherwise be incompatible withthe existence of the crystal lattice or even with the existence ofintact protein.

[0100] Advantageously, crosslinking according to the present inventionis carried out in such a way that, under conditions of storage, thecrosslinking interactions prevent the constituent protein molecules inthe crystal from going back into solution, effectively insolubilizing orimmobilizing the protein molecules into microcrystalline particles. Uponexposure to a trigger in the environment surrounding the crosslinkedprotein crystals, such as under conditions of use rather than storage,the protein molecules dissolve, releasing their protein activity. Therate of dissolution is controlled by one or more of the followingfactors: the degree of crosslinking, the length of time of exposure ofprotein crystals to the crosslinking agent, the amino acid residuesinvolved in the crosslinks, whether the crosslinker is homobifunctionalor heterobifunctional, the rate of addition of crosslinking agent to theprotein crystals, the nature of the crosslinker, the chain length of thecrosslinker, the surface area of the crosslinked protein crystals, thesize of the crosslinked protein crystals, the shape of the crosslinkedprotein crystals and combinations thereof.

[0101] Alternatively, controlled dissolution crosslinked proteincrystals function as crosslinked protein crystals in the absence of thespecific trigger required for initiating and maintaining dissolution.

[0102] Crosslinking can be achieved using one or a combination of a widevariety of multifunctional reagents, at the same time (in parallel) orin sequence, including bifunctional reagents. Upon exposure to a triggerin the surrounding environment, or over a given period of time, thecrosslinks between protein crystals crosslinked with suchmultifunctional crosslinking agents lessen or weaken, leading to proteindissolution or release of activity. Such crosslinking agents includeglutaraldehyde, succinaldehyde, octanedialdehyde and glyoxal. Additionalmultifunctional crosslinking agents include halo-triazines, e.g.,cyanuric chloride; halo-pyrimidines, e.g.,2,4,6-trichloro/bromo-pyrimidine; anhydrides or halides of aliphatic oraromatic mono- or di-carboxylic acids, e.g., maleic anhydride,(meth)acryloyl chloride, chloroacetyl chloride; N-methylol compounds,e.g., N-methylol-chloro acetamide; di-isocyanates or di-isothiocyanates,e.g., phenylene-1,4-di-isocyanate and aziridines. Other crosslinkingagents include epoxides, such as, for example, di-epoxides, tri-epoxidesand tetra-epoxides. According to a preferred embodiment of thisinvention, the crosslinking agent is glutaraldehyde, used alone or insequence with an epoxide. For a representative listing of otheravailable crosslinking reagents see, for example, the 1996 catalog ofthe Pierce Chemical Company. Such multifunctional crosslinking agentsmay also be used, at the same time (in parallel) or in sequence, withreversible crbsslinking agents, such as those described below.

[0103] Crosslinkers useful in various embodiments of this invention are(1) those which create covalent links from one cysteine side chain of aprotein to another another cysteine side chain, (2) those which createcovalent links from one lysine side chain of a protein to another, or(3) those which create covalent links from one cysteine side chain of aprotein to a lysine side chain. Crosslinking may occur throughintermolecular and intramolecular covalent crosslinks.

[0104] Protein crystals may be crosslinked with one of the followingcrosslinkers to produce controlled dissolution crosslinked proteincrystals: dimethyl 3, 3′-dithiobispropionimidate.HCl (DTBP); dithiobis(succinimidylpropionate) (DSP); bismaleimidohexane (BMH);bis[Sulfosuccinimidyl]suberate (BS); 1,5-difluoro-2,4-dinitrobenzene(DFDNB); dimethylsuberimidate.2HCl (DMS); disuccinimidyl glutarate(DSG); disulfosuccinimidyl tartarate (Sulfo-DST);1-ethyl-3-[3-dimethylaminoproplyl]carbodiimide hydrochloride (EDC);ethylene glycolbis [sulfo-succinimidylsuccinate] (Sulfo-EGS);N-[γ-maleimido-butyryloxy]succinimide ester (GMBS);N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB);sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate(Sulfo-LC-SMPT); bis-[β-(4-azidosalicylamido) ethyl]disulfide (BASED);and NHS-PEG-Vinylsulfone (NHS-PEG-VS).

[0105] According to an alternate embodiment of this invention,crosslinking may be carried out using reversible crosslinkers, inparallel or in sequence. The resulting crosslinked protein crystals arecharacterized by a reactive multi-functional linker, into which atrigger is incorporated as a separate group. The reactive functionalityis involved in linking together reactive amino acid side chains in aprotein and the trigger consists of a bond that can be broken byaltering one or more conditions in the surrounding environment (e.g.,pH, temperature, or thermodynamic water activity). This is illustrateddiagrammatically as:

[0106] X-Y-Z+2 AA residues -->AA₁-X-Y-Z-AA₂

[0107] change in environment -->AA₁-X+Y-Z-AA₂

[0108] where X and Z are groups with reactive functionality

[0109] where Y is a trigger

[0110] where AA₁ and AA₂ represent reactive amino acid residues on thesame protein or on two different proteins. The bond between thecrosslinking agent and the protein may be a covalent or ionic bond, or ahydrogen bond. The change in surrounding environment results in breakingof the trigger bond and dissolution of the protein. Thus, the crosslinksbetween protein crystals crosslinked with such reversible crosslinkingagents break, leading to protein crystal dissolution or release ofactivity.

[0111] Alternatively, the reactive functionality of the crosslinker andthe trigger may be the same, as in:

[0112] X-Z+2AA residues -->AA₁-X-Z-AA₂

[0113] change in environment -->AA₁+X-Z-AA₂

[0114] The crosslinker may be homofunctional (X=Y) or heterofunctional(X is not equal to Y). The reactive functionality X and Y may be, butnot limited to the following functional groups (where R, R′, R″, and R′″may be alkyl, aryl or hydrogen groups):

[0115] I. Reactive acyl donors are exemplified by: carboxylate estersRCOOR′, amides RCONHR′, Acyl azides RCON₃, carbodiimides R—N═C═N—R′,N-hydroxyimide esters, RCO—O—NR′, imidoesters R—C═NH2⁺ (OR′), anhydridesRCO—O—COR′, carbonates RO—CO—O—R′, urethanes RNHCONHR′, acid halidesRCOHal (where Hal=a halogen), acyl hydrazides RCONNR′R″, O-acylisoureasRCO—O—C═NR′ (—NR″R′″),

[0116] II. Reactive carbonyl groups are exemplified by: aldehydes RCHOand ketones RCOR′, acetals RCO(H₂)R′, ketals RR′CO₂R′R″. Reactivecarbonyl containing functional groups known to those well skilled in theart of protein immobilization and crosslinking are described in theliterature [Pierce Catalog and Handbook, Pierce Chemical Company,Rockford, Ill. (1994); S. S. Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press, Boca Raton, Fla. (1991)].

[0117] III. Alkyl or aryl donors are exemplified by: alkyl or arylhalides R-Hal, azides R—N₃, sulfate esters RSO₃R′, phosphate estersRPO(OR′₃), alkyloxonium salts R₃O+, sulfonium R₃S+, nitrate estersRONO₂, Michael acceptors RCR′═CR′″COR″, aryl fluorides ArF, isonitrilesRN+═C—, haloamines R₂N-Hal, alkenes and alkynes.

[0118] IV. Sulfur containing groups are exemplified by disulfides RSSR′,sulfhydryls RSH, epoxides R₂C ^(O) CR′₂.

[0119] V. Salts are exemplified by alkyl or aryl ammonium salts R₄N+,carboxylate RCOO—, sulfate ROSO₃—, phosphate ROPO₃″ and amines R₃N.

[0120] The table below includes examples of triggers, organized byrelease mechanism. In the table, R═ is a multifunctional crosslinkingagent that can be an alkyl, aryl, or other chains with activating groupsthat can react with the protein to be crosslinked. Those reactive groupscan be any variety of groups such as those susceptible to nucleophilic,free radical or electrophilic displacement including halides, aldehydes,carbonates, urethanes, xanthanes, epoxides among others. Release TriggerExamples Conditions 1. Acid Labile R—O—R H⁺ or Lewis Linkers e.g. Thp,MOM, Acidic catalysts Acetal, ketal Aldol, Michael adducts, esters 2.Base Labile R'OCO2—R' Variety of basic Linkers Carbonates mediaR'O—CONR₂ Carbamates R₂'NCONR₂ Urethanes Aldol, Michael adducts, esters3. Fluoride R—OSiR₃ Aqueous F⁻ Labile Linkers Various Si containinglinkers 4. Enzyme RCOOR, RCONR₂' Free lipases, Labile Linkers amidases,esterases 5. Reduction Disulfide H₂ catalyst; Labile Linkers linkersthat Hydrides cleave via Hydrogenolysis Reductive Elimination R'—S—S—R6. Oxidation R—OSiR₃ Oxidizing Labile Linkers Glycols R— agents: e.g. CH(OH)—CH (OH)—R' H₂O₂, NaOCl, IO₄ ⁻ Metal based oxidizers, otherhypervalent oxidents 7. Thio-labile R'—S—S—R Thiols, e.g., linkers Cys,DTT, mercaptoethanol 8. Heavy Metal Various Allyl Transition metalLabile Linkers Ethers based reagents ROCH₂CH═CHR (Pd, Ir, Hg, Ag, Alkyl,Acyl Cu, Tl, Rh) Allyl ester Pd (0) catalysts 9. PhotolabileO-nitrobenzyl light (hv) Linkers (ONB) DESYL groups in linker 10. FreeThiohydroxamate Free radical Radical Labile ester initiator Linkers(Barton ester) 11. Metal- Iron (III) Metal removal chelate linkeddiphenanthroline e.g. by chelation or precipitation 12. ThermallyPeroxides Increase in Labile Linkers R—OO—R temperature 13. “SafetyMethylthio- Base; amines, Catch” Labile ethyl (Mte) others LinkersDithianes

[0121] The dissolution of the crosslinked protein crystals of thisinvention can be controlled by selecting appropriate crosslinkers andthe associated triggers. Examples of physical and chemical triggersavailable include: change in temperature, change in pH, change inchemical composition, change from concentrate to dilute form, change inoxidation-reduction potential of the solution, change in the incidentradiation, change in transition metal concentration, change in flourideconcentration, change in free radical concentration, change in metalchelater concentration, change in shear force acting upon the crystaland combinations thereof.

[0122] Additional examples of reversible crosslinkers are described inT. W. Green, Protective Groups in Organic Synthesis, John Wiley & Sons(Eds.) (1981). Any variety of strategies used for reversible protectinggroups can be incorporated into a crosslinker suitable for producingcrosslinked protein crystals capable of reversible, controlledsolubilization. Various approaches are listed, in Waldmann's review ofthis subject, in Angewante Chemie Inl. Ed. Engl., 35, p. 2056 (1996).

[0123] Other types of reversible crosslinkers are disulfidebond-containing crosslinkers. The trigger breaking crosslinks formed bysuch crosslinkers is the addition of reducing agent, such as cysteine,to the environment of the crosslinked protein crystals.

[0124] Disulfide crosslinkers are described in the Pierce Catalog andHandbook (1994-1995).

[0125] Examples of such crosslinkers include:

[0126] Homobifunctional (Symmetric)

[0127] DSP—Dithiobis(succinimidylpropionate), also know as Lomant,'sReagent

[0128] DTSSP—3-3′-Dithiobis(sulfosuccinimidylpropionate), water solubleversion of DSP

[0129] DTBP—Dimethyl 3,3′-dithiobispropionimidate-HCl

[0130] BASED—Bis-(β-[4-azidosalicylamido]ethyl)disulfide

[0131] DPDPB—1,4-Di-(3′-[2′-pyridyldithio]-propionamido)butane.

[0132] Heterobifunctional (Asymmetric)

[0133] SPDP—N-Succinimidyl-3-(2-pyridyldithio)propionate

[0134] LC-SPDP—Succinimidyl-6-(3-[2-pyridyldithio] propionate)hexanoate

[0135] Sulfo-LC-SPDP—Sulfosuccinimidyl-6-(3-[2-pyridyldlthio]propionate)hexanoate, water soluble version of LC-SPDP

[0136] APDP—N-(4-[p-azidosalicylamido]butyl)-3′-(2′-pyridyldithio)propionamide

[0137] SADP—N-Succinimidyl(4-azidophenyl)1,3′-dithiopropionate

[0138] Sulfo-SADP—Sulfosuccinimidyl(4-azidophenyl)1,3′-dithiopropionate, water soluble version of SADP

[0139]SAED—Sulfosuccinimidyl-2-(7-azido-4-methycoumarin-3-acetamide)ethyl-1,3′dithiopropionate

[0140]SAND—Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3′-dithiopropionate

[0141]SASD—Sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate

[0142] SMPB—Succinimidyl-4-(p-maleimidophenyl)butyrate

[0143] Sulfo-SMPB—Sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate

[0144] SMPT—4-Succinimidyloxycarbonyl-methyl-α-(2-pyridylthio) toluene

[0145]Sulfo-LC-SMPT—Sulfosuccinimidyl-6-(α-methyl-α-(2-pyridylthio)toluamido)hexanoate.

[0146] In order that this invention may be better understood, thefollowing examples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any manner.

EXAMPLES Example 1 Preparation of Crosslinked Subtilisin Crystals

[0147] Crystallization of Subtilisin

[0148] One volume of Alcalase 2.5L (Novo Nordisk Bioindustrials,Franklinton, N.C.) was added to 2 volumes of a solution of 15% sodiumsulfate (pH 5.5) prepared at 30-35° C. The crystallization solution wasseeded with {fraction (1/2,000)}-{fraction (1/500)} volume seeds (30mg/ml slurry of crystals in 15% sodium sulfate (pH 5.5), pH supported at5.5 by adding NaOH. The seeded crystallization solution was incubated at30-35° C., stirring by magnetic stirrer overnight. This yielded 60-80%(by activity) crystal rods, 10-50 μm, in length, 1-3 μm in width, after24-48 hours.

Example 2 Crosslinking of Subtilisin Crystals

[0149] Subtilisin crystals were crosslinked using one of a variety ofcrosslinkers, including: glutaraldehyde, glyoxal, succinaldehyde,octanedialdehyde and epoxides.

[0150] Glutaraldehyde Crosslinking

[0151] Glutaraldehyde (“GA”) (supplied as 50% in aqueous by AldrichChemical Co.) was diluted in deionized water at 4° C. in the variousamounts listed in Table I below. For each ml of subtilisin crystals (27mg/ml) in 15% sodium sulfate, 10 μl of the diluted glutaraldehyde wasadded to the slurry while shaking on a vortex at low speed (for amountsless than 5 ml) or stirring with an overhead stirrer at medium speed(for amounts 25 ml-500 ml). After mixing for the allotted crosslinkingtime, the samples were centrifuged for 20 seconds at maximum speed, thesupernatant was discarded and replaced with 15% sodium sulfate. This“washing” was repeated a total of 5 times. The final resuspension waseffected with 900 μl of 15% sodium sulfate. TABLE I GlutaraldehydeCrosslinking Crosslinking % GA - final GA (ml) H₂O (ml) time (min)0.0076 1.0 64.96 60 0.0189 1.0 25.46 39 0.02 1.0 24.0 39, 81 0.05 1.09.00 15, 60, 89 0.08 1.0 5.25 39, 81 0.10 1.0 4.00 60, 81 0.125 1.0 3.003, 10, 17, 39 0.15 1.0 2.33 81, 120 0.20 1.0 1.50 19, 60, 120 0.231 1.01.16 10, 39, 120 0.3 1.0 0.67 60 0.5 1.0 0 60

[0152] Glyoxal Crosslinking

[0153] Glyoxal (supplied as 40% in aqueous by Aldrich Chemical Co.)(“GY”) was added to the crystal suspension to give a final concentrationof 0.01-1.0%. For each ml of subtilisin crystals (27 mg/ml) in 15%sodium sulfate 0.25 μl to 25 ml (0.01 to 1%) of the glyoxal was added tothe slurry, while magnetically stirring at ambient temperature. Afterstirring for 1 hour, the crosslinked crystals were centrifuged andwashed, as described for glutaraldehyde crosslinking.

[0154] Octanedialdehyde Crosslinking

[0155] Octanedialdehyde (“OA”) (100% as supplied by DSM Chemie Linz), inthe amounts shown in Table II below, was added undiluted to 1 ml ofsubtilisin crystal slurry (27 mg/ml in 15% sodium sulfate) whilemagnetically stirring at ambient temperature. Stirring was continued forthe specified time of minutes or hours before the crosslinked crystalswere centrifuged and washed, as described for glutaraldehydecrosslinking. TABLE II Octanedialdehyde Crosslinking % OA - final OA(μl) Crosslinking time 0.05 0.5 16 h 0.1 1.0 16 h 0.2 2.0 16 h 0.25 2.516 h 0.5 5.0 16 h 1.0 10.0 30 m, 1 h, 3 h, 16 h

[0156] Succinaldehyde Crosslinking

[0157] Succinaldehyde (“SA”)(40% as supplied by DSM Chemie Linz) wasadded undiluted, in the amounts shown in Table III below, to 1 ml ofsubtilisin crystal slurry (27 mg/ml in 15% sodium sulfate) whilemagnetically stirring at ambient temperature. Stirring was continued forthe specified time of minutes or hours before the crosslinked crystalswere centrifuged and washed, as described for glutaraldehydecrosslinking. TABLE III Succinaldehyde Crosslinking % SA - final SA (μl)Crosslinking time 1.0 25 30 m, 1 h, 3 h

[0158] Epichlorohydrin Crosslinking

[0159] A 10 μl aliquot of epichlorohydrin (“EP”) (99%, Sigma ChemicalCo., St. Louis, Mo.) was added undiluted to 1 ml of subtilisin crystalslurry (27 mg/ml in 15% sodium sulfate) while stirring at ambienttemperature. Stirring was continued for the specified time of minutes orhours before the crosslinked crystals were centrifuged and washed, asdescribed for glutaraldehyde crosslinking.

[0160] Epoxide Crosslinking

[0161] General Procedure

[0162] Crosslinking of subtilisin was carried out individually using oneof a variety of epoxides. These included:

[0163] 1) General name—DENACOL

[0164] a) DENACOL EX-411

[0165] b) DENACOL EX-421

[0166] c) DENACOL EX-614

[0167] d) DENACOL EX-201

[0168] e) DENACOL EX-202; all obtained from Nagase American Corporation.

[0169] 2) Obtained from Tokyo Kasei Inc. America:

[0170] a) Neopentyl Glycol diglycidyl Ether (N448)(“NP”)

[0171] b) Ethylene Glycol diglycidyl Ether (EO342)(“EG”).

[0172] The concentration of the epoxide was varied between 0.01 and 4.0%and the crosslinking time was varied from 1 hour to 72 hours. Theprocedure for addition to and removal of crosslinker from enzyme was asdescribed above for glutaraldehyde crosslinking.

[0173] Subsequent crosslinking with glutaraldehyde (0.01 to 0.2%) for (1hour to 5 hours) yielded strongly crosslinked enzyme crystals, insolublein water, but active in the azocasein assay.

[0174] A sample of 1 ml of subtilisin crystal slurry (27 mg/ml in 15%sodium sulfate) was mixed by vortexing at low speed to assure a uniformsuspension of crystals. Epoxide (10% solution in DMF) was added to thecrystal slurry in the amounts specified in Table IV, and the mixture wasshaken at ambient temperature. After the allotted time between 1 and 72hours at ambient temperature, glutaraldehyde (10% in DMF) was added tothe epoxide/crystal mixture and stirring was continued at ambienttemperature for the time specified in Table IV. The resultingcrosslinked enzyme crystals were washed 2×with 1% (NH₄)₂SO₄/10 mM CaCl₂then 3×with water and finally 1×with 1% (NH₄)₂SO₄/10 mM CaCl₂ beforeresuspending in 1% (NH₄)₂SO₄/10 mM CaCl₂. TABLE IVEpoxide/Glutaraldehyde Crosslinking Epoxide Glutaraldehyde EpoxideEpoxide Crosslinking Glutaraldehyde Crosslinking Name Amount Time AmountTime EX-411 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h EX-421 0.01-4% 1-72 h0.01-0.1% 0.5-2 h EX-614 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h EX-201 0.01-4%1-72 h 0.01-0.1% 0.5-2 h EX-202 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h NP0.01-4% 1-72 h 0.01-0.1% 0.5-2 h (N448) EG 0.01-4% 1-72 h 0.01-0.1%0.5-2 h (EO342)

[0175] Large Scale Preparation of A Preferred Epoxide Sample

[0176] Prior to crosslinking, a sample of 380 ml of crystallinesubtilisin in 15% sodium sulfate (27 mg/ml) was mixed by overheadstirring at ambient temperature for 5 minutes to assure a uniformsuspension of crystals. Neopentyl glycol diglycidyl ether (3.838 ml of a10% solution in DMF) was added to the crystal slurry and the mixture wasstirred at ambient temperature. After 5 hours at ambient temperature,3.838 ml of glutaraldehyde (10% in DMF) was added to the epoxide/crystalmixture and stirring was continued at ambient temperature for 1.5 hours.The resulting crosslinked enzyme crystals were washed 2×with 1%(NH₄)₂SO₄/10 mM CaCl₂ then 3×with water and finally 1×with 1%(NH₄)₂SO₄/10 mM CaCl₂, before resuspending in 1% (NH₄)₂SO₄/10 mM CaCl₂.

Example 3 Activity Assay

[0177] In order to test the activity of crosslinked protein crystalsaccording to this invention, as well as other enzyme samples, wedeveloped the following azocasein assay.

[0178] Materials:

[0179] 2.0M Tris Buffer. 500 ppm CaCl₂

[0180] 0.2M Tris Buffer. 50 ppm CaCl₂

[0181] 50% urea

[0182] Azocasein

[0183] 5% trichloroacetic acid (“TCA”)

[0184] Alcalase (2.5L)

[0185] ChiroCLEC-BL™ (crosslinked subtilisin crystals, available fromAltus Biologics, Inc., Cambridge, Mass.)

[0186] The assay was carried out, preparing azocasein just prior to use,by dissolving 600 mg azocasein with 10 ml of 50% urea and vortexinglightly to complete the dissolution. Then 10 ml 2.0M Tris was added andvortexed to mix, increasing the volume to 100 ml by adding deionizedwater.

[0187] The stock solutions of the enzyme to be assayed in 0.2M Tris wereprepared, to provide 50 μl aliquots to be assayed, as follows:

[0188] Without detergent: 0.03 mg/ml Alcalase (soluble,

[0189] uncrosslinked subtilisin

[0190] Carlsberg 80.3 mg/ml)

[0191] 3.0 mg/ml ChiroCLEC-BL™.

[0192] With 120 μl detergent/ml solution:

[0193] 0.03 mg/ml Alcalase

[0194] 3.0 mg/ml ChiroCLEC-BL™.

[0195] We added 50 μl aliquots of enzyme to 150 μl of 0.2M Tris andplaced the mixtures in 5 ml test tubes with micro-stir bars. We thenwarmed both the test tubes and the azocasein at 40° C. for 1 minuteusing a metal heating block. After that, we added 1 ml of the azocaseinto each tube and stirred at 40° C. for 15 minutes using the heatingblock at stir speed 4. We then added 2 ml TCA to each tube, mixing byvortex, and placed the tubes in an ice bath immediately, allowing thesamples to stand at 0° C. for 20 minutes. We microfuged the samples for5 minutes at maximum rpm and microfiltered, if necessary. We measuredabsorbance of the expressed activity in abs-units/mg protein.minsupernatant at λ390. In this assay, all measurements were done intriplicate. Controls were void of enzyme but contained detergent if itwas present in the assay. This time=0 assay was repeated at time=15minutes and other times, if necessary.

[0196] The detergents used in the various assays included Tide, Wisk andCiba-Geigy detergents #15, #16 and #44 (“Ciba detergents”). Cibadetergent #15 constitutes a typical European detergentformulation—liquid (aqueous) detergent on the basis of 15% alkylbenzenesulfonate, 14% fatty alcohol ethoxylate and 10% fatty acid salt (soap).Ciba detergent #16 constitutes a typical United States detergentformulation—liquid (aqueous) detergent on the basis of 7.5% alkylbenzene sulfonate, 10% fatty alcohol ethoxylate and 17% alkyl eithersulfate. Ciba detergent #44 constitutes a typical compact detergentformulation—liquid (aqueous) detergent on the basis of 6% fatty alcoholethoxylate, 23% alkyl ether sulfate and 10% sodium citrate. Cibadetergents #15, #16 and #44 may be obtained upon request from CibaSpecialty Chemicals Corp., Division Consumer Care Chemicals, Greensboro,N.C.

[0197] We prepared assay stock solutions from dilution stocks, andcarried out the assays, as follows.

[0198] Activity Assay—200×Dilution—Crosslinked Subtilisin Crystals andCrystalline Subtilisin in Heavy Duty Liquid Detergent (Ciba #15, Ciba#44, Tide and Wisk)

[0199] Stocks A, B, C and D were prepared in 10 ml neoprene tubes asfollows.

[0200] Stock A: Crosslinked Subtilisin Crystals Prepared According tothis Invention (−27 mg/ml)

[0201] We centrifuged 37 μl slurry of crosslinked subtilisin crystals(equal to 1 mg crosslinked enzyme crystals) to remove supernatant, added1 ml detergent and vortexed to mix. A 50 μl aliquot of the resultingmixture was added to 9.95 ml water, to a final concentration of 5 μg/ml.

[0202] Stock B: Uncrosslinked Subtilisin Crystals (˜27 mg/ml)

[0203] We centrifuged 37 μl slurry of subtilisin crystals (equal to 1 mgenzyme crystals) to remove supernatant, added 1 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to9.95 ml water, to a final concentration of 5 μg/ml.

[0204] Stock C: Alcalase

[0205] We added 18.75 μl Alcalase (80.3 mg/ml) to 3 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to9.95 ml water, to a final concentration of 2.5 μg/ml.

[0206] Stock D: Detergent

[0207] A 50 μl aliquot of detergent was added to 9.95 ml water.

[0208] Azocasein stock (6 mg/ml) was prepared as described above. Upondilution of Stock A and B to 5 μg/ml, the t=0 assay was set upimmediately and carried out as described above, except that the amountof stock sample used was 200 μl, instead of 50 μl+150 μl 0.2M Tris.While the tubes were heating for 1 minute at 40° C., two additionalsamples of 2 ml each of Stocks A, B and C were placed in 1.5 mlmicrocentrifuge tubes and heated to 52° C. while shaking for furthertesting after 5 minutes and 15 minutes dilution with heating.

[0209] Activity Assay—670×Dilution—Crosslinked Subtilisin Crystals andCrystalline Subtilisin in Detergent Concentrate (Ciba #16)

[0210] Stocks A, B, C and D were prepared in 10 ml neoprene tubes asfollows.

[0211] Stock A: Crosslinked Subtilisin Crystals Prepared Accordinq tothis Invention (˜27 mg/ml)

[0212] We centrifuged 124 μl slurry of crosslinked subtilisin crystals(equal to 3.35 mg crosslinked enzyme crystals) to remove supernatant,added 1 ml detergent and vortexed to mix. A 50 μl aliquot of theresulting mixture was added to 33.45 ml water, to a final concentrationof 5 μg/ml.

[0213] Stock B: Uncrosslinked Subtilisin Crystals (˜27 mg/ml)

[0214] We centrifuged 124 μl slurry of subtilisin crystals (equal to3.35 mg enzyme crystals) to remove supernatant, added 1 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to33.45 ml water, to a final concentration of 5 μg/ml.

[0215] Stock C: Alcalase

[0216] We added 167 μl Alcalase (80.3 mg/ml) to 8 ml detergent andvortexed to mix. A 50 μl aliquot of the resulting mixture was added to33.45 ml water, to a final concentration of 2.5 μg/ml.

[0217] Stock D: Detergent

[0218] A 50 μl aliquot of detergent was added to 33.45 ml water.

[0219] Azocasein stock (6 mg/ml) was prepared as described above. Thet=0 assay was set up immediately and carried out as described above,except that the amount of stock sample used was 200 μl, instead of 50μl, plus 150 μl 0.2M Tris buffer. While the tubes were heating for 1minute at 40° C., two additional samples of 2 ml each of Stocks A, B andC were placed in Eppendorf tubes and heated to 40° C. while shaking forfurther testing after 5 minutes and 15 minutes dilution with heating.

Example 4 Stability Study

[0220] In order to test the stability of crosslinked enzyme crystalsaccording to this invention, as well as other enzyme samples, wedeveloped the following assays.

[0221] Azocasein Assay—Stability Study 52° C.

[0222] First, we prepared stock solutions of the enzyme samples indetergent in 2 ml Eppendorf tubes with screw caps. After incubating themixtures in a water bath at 52° C. for the appropriate times, we added1.47 ml of 0.2M Tris buffer to one of each enzyme sample tube, and mixedwell. To assay for activity after the appropriate time of incubationfollowed by dilution, we removed a 50 μl aliquot from each tube andassayed as described below. The remaining samples of enzyme/detergentstocks were placed in a water bath at 52° C., with further aliquotsbeing removed for assay at specific times.

[0223] The assay was performed by adding 50 μl enzyme sample to 150 μl0.2M Tris buffer and heating to 40° C. for 1 minute. At a constant 40°C. temperature, we then added 1.0 ml azocasein stock (as described inExample 1) to each sample, stirring for 15 minutes using a heating blockat stir speed 4. We then added 2 ml TCA to each tube, mixing by vortex,and placed the tubes in an-ice bath immediately, allowing the samples tostand at 0° C. for 20 minutes. We microfuged the samples for 5 minutesat maximum rpm and microfiltered, if necessary. We measured absorbanceof the supernatant at λ390 and expressed activity as abs-units/mgprotein.min. In this assay, all measurements were done in triplicate.Controls were void of enzyme but contained detergent if it waspresent-in the assay.

[0224] In order to assess activity as part of stability studies carriedout at 52° C., we prepared assay stock solutions from dilution stocks,and carried out the assays, as follows.

[0225] For Alcalase

[0226] Stocks:

[0227] Stock A: Alcalase (80.3 mg/ml) in commercial detergent (Tide orWisk, deactivated by heating at 70° C. for 4 hours)−finalconcentration=0.25 mg/ml. The stock was prepared by adding 31.2 μlAlcalase to 9.97 ml detergent. A 200 μl aliquot of the resulting mixturewas placed in each of several 2 ml Eppendorf tubes (3×for t=0, 30 andothers)

[0228] Stock B: Alcalase (80.3 mg/ml) in Ciba detergent (Ciba #15, Ciba#16 or Ciba #44)−final concentration=0.25 mg/ml. The stock was preparedby adding 31.2 μl Alcalase to 9.97 ml detergent. A 200 #l aliquot of theresulting mixture was placed in each of several 2 ml Eppendorf tubes (3×for t=0, 1 hour and 4-6 hours).

[0229] Stock C: Commercial detergent (Tide or Wisk, deactivated byheating at 70° C. for 4 hours) 3×(200 μl of the above in 2 ml Eppendorftubes).

[0230] Stock D: Ciba detergent (Ciba #15, Ciba #16 or Ciba #44) 3×(200μl of the above in 2 ml Eppendorf tubes).

[0231] The t=0 assay was performed immediately after 1.47 ml of 0.2 MTris was added to one of each of tubes containing Stocks A-D and thecontents mixed well. Remaining samples of Stocks A-D were placed in awater bath and heated to 52° C. Otherwise, the assays were carried outas described above.

[0232] For Crosslinked Subtilisin

[0233] Crystals and Crystalline Subtilisin

[0234] Stocks A, B, C and D were prepared in 2 ml Eppendorf tubes withscrew caps as follows.

[0235] Stock A: ChiroCLEC-BL™ in commercial detergent (denatured Tide orWisk)−final concentration=25 mg/ml. The stock was prepared bycentrifuging 3.12 ml enzyme slurry to remove water and then diluting theenzyme to 10 ml with detergent. A 200 μl aliquot of the resultingmixture was placed in each of several 2 ml Eppendorf tubes (4×for t=0,24 hours, 48 hours and 72 hours).

[0236] Stock B: ChiroCLEC-BL™ in Ciba detergent (Ciba #15, Ciba #16 orCiba #44)−final concentration=25 mg/ml. The stock was prepared bycentrifuging 3.12 ml enzyme slurry to remove water and then diluting theenzyme to 10 ml with detergent. A 200 μl aliquot of the resultingmixture was placed in each of several 2 ml Eppendorf tubes (4×for t=0,24 hours, 48 hours and 72 hours at 52° C.).

[0237] Stock C: Commercial detergent (Tide or Wisk, deactivated byheating at 70° C. for 4 hours) 4×(200 μl of the above in 2 ml Eppendorftubes).

[0238] Stock D: Ciba-Geigy detergent (Ciba #15, #16 or #44, depending onwhich detergent was chosen for Stock B) 4×(200 μl of the above in 2 mlEppendorf tubes).

[0239] The t=0 assay was set up immediately after 1.47 ml of 0.2M Triswas added to one of each of tubes containing Stocks A-D and the contentsmixed well. Remaining samples of Stocks A-D were placed in a water bathand heated to 52° C. Otherwise, the assays were carried out as describedabove.

[0240] For Crosslinked Subtilisin Crystals and Crystalline Subtilisin

[0241] Stocks A, B and C were prepared in 2 ml Eppendorf tubes withscrew caps as follows.

[0242] Stock A: Uncrosslinked subtilisin crystals (˜27 mg/ml) in Cibadetergent (Ciba #15, #16 or #44)−final concentration=˜1 mg/ml. The stockwas prepared by centrifuging 50 μl crystal slurry to remove supernatant,then adding 1.35 ml detergent (Ciba #15, Ciba #16 or Ciba #44) to afinal concentration of 1 mg/ml. An 80 μl aliquot of the resultingmixture was placed in each of several 2.0 ml Eppendorf tubes (3×for t=0,15 minutes and others).

[0243] Stock B: Crosslinked subtilisin crystals according to thisinvention (˜27 mg/ml) in Ciba detergent (Ciba #15, #16 or #44)−finalconcentration=˜1 mg/ml. The stock was prepared by centrifuging 50 μlcrystal slurry to remove supernatant, then adding 1.35 ml detergent, toa final concentration of 1 mg/ml. An 80 μl aliquot of the resultingmixture was placed in each of several 2.0 ml Eppendorf tubes (3×for t=0,15 minutes and others).

[0244] Stock C: Ciba detergent (Ciba #15, #16 or #44)−3×(80 μl of theabove in 2.0 ml tubes).

[0245] The t=0 assay was set up immediately after 1.8 ml of water wasadded to one of each of tubes containing Stocks A-C and the contentsmixed well. Remaining samples of Stocks A-C were placed in a water bathand heated to 52° C. Otherwise, the assays were carried out as describedabove, except for the addition of 150 μl 0.2 M Tris buffer instead of200 μl.

[0246] Azocasein Assay—Stability Study 40° C.

[0247] First, we prepared stock solutions of the enzyme samples indetergent in 2 ml Eppendorf tubes with screw caps. To assay stability att=0, we added 1.8 ml of deionized water to a 25 μl of each sample andmixed well. We removed a 25 μl aliquot from each tube and assayed asdescribed below. The remaining samples of enzyme/detergent stocks wereplaced in a water bath at 40° C., with further aliquots being removedfor assay at specific times.

[0248] The assay was performed by adding 25 al of the diluted enzymesample to 175 μl 0.2M Tris buffer and heating to 40° C. for 1 minute. Ata constant 40° C. temperature, we then added 1.0 ml azocasein stock (asdescribed in Example 3) to each sample, stirring for 15 minutes using aheating block at stir speed 4. We then added 2 ml TCA to each tube,mixing by vortex, and placed the tubes in an ice bath immediately,allowing the samples to stand at 0° C. for 20 minutes. We microfuged thesamples for 5 minutes at maximum rpm and microfiltered, if necessary. Wemeasured absorbance of the supernatant at λ390 and expressed activity asabs.units/mg protein.min. In this assay, all measurements were done intriplicate. Controls were void of enzyme but contained detergent if itwas present in the assay.

[0249] In order to assess activity as part of stability studies carriedout at 40° C., we prepared assay stock solutions from dilution stocks,and carried out the assays, as follows.

[0250] Stock A: Crosslinked Subtilisin Crystals According to thisInvention (˜27 mg/ml)

[0251] We centrifuged 124 μl slurry of crosslinked subtilisin crystals(equal to 3.35 mg crosslinked enzyme crystals) to remove supernatant,added 1 ml detergent and vortexed to mix, to a final concentration of3.35 mg/ml.

[0252] Stock B: Uncrosslinked Subtilisin Crystals (˜27 mg/ml)

[0253] We centrifuged 124 μl slurry of subtilisin crystals (equal to3.35 mg enzyme crystals) to remove supernatant, added 1 ml detergent andvortexed to mix, to a final concentration of 3.35 mg/ml.

[0254] Stock C: Alcalase

[0255] We added 20.9 μl Alcalase (80.3 mg/ml) to 1 ml Ciba detergent(Ciba #15, Ciba #16 or Ciba #44) and commercial detergent (Tide or Wisk,denatured by heating at 70° C. for 4 hours) and vortexed to mix, to afinal concentration of 1.67 mg/ml.

[0256] Stock D: Detergent

[0257] One ml of commercial detergent (Tide or Wisk, deactivated byheating at 70° C. for 4 hours) and Ciba detergents #15, #16 and #44. A25 μl aliquot of each stock was added to 1.8 ml of deionized water andmixed well. A further 25 μl aliquot of the diluted stock was added toeach reaction tube.

[0258] The t=0 assay was performed immediately after 175 μl of 0.2M Triswas added to each tube containing 25 μl of the various stock samples.Remaining samples of Stocks A-D were placed in a water bath and heatedto 40° C. Otherwise, the assays were carried out as described above.

Example 5 Dissolution Studies

[0259] We also assessed the characteristics of crosslinked enzymecrystals according to this invention, as well as other enzyme samples,with respect to dissolution in concentrate and upon dilution, asdetailed below. Stock solutions were prepared and diluted as describedabove. The resulting dispersions were heated at 40° C. and analyzedunder a microscope at 250×for dissolution progress.

Example 6 Results of Activity and Stability Assays

[0260] Crosslinked enzyme crystals of subtilisin, as described above, aswell as soluble enzymes and other commercial enzymes, alone and in thepresence of commercial detergents, were tested for activity in theazocasein assay, as described above. Catalyst concentrations forequivalent activities were determined for Alcalase, ChiroCLEC-BL™, Wiskwith active protease and Tide with active protease:

[0261] ChiroCLEC-BL™: 150 μg/6 μl detergent 0.4-0.5 absorbance units

[0262] Alcalase: 15 μg/6 μl detergent 0.5-0.6 absorbance units

[0263] Tide: 6 μl detergent approximately 0.6 absorbance units

[0264] Wisk: 6 μl detergent approximately 0.6 absorbance units.

[0265] The dilution studies (discussed supra) were started by assessingthe activities of A-lcalase and uncrosslinked crystals of Alcalase inCiba detergents #15 and #16. Initial activities were comparable andlosses of up to ˜50% were seen after 15 minutes at 52° C.

[0266] Table V summarizes the stability of samples of Alcalase (0.25mg/ml) and ChiroCLEC-BL™ (25 mg/ml) in denatured Wisk or Tide detergent,or in Ciba detergents #15 and #16 at 52° C. Activity was measured by theazocasein assay. TABLE V Stability of Subtilisin in Detergents at 52° C.Alcalase T_(1/2) ChiroCLEC-BL ™ T_(1/2) Detergent at 52° C. at 52° C.#15 less than 15 min >>100 hours #16 less than 15 min >>100 hours Tide(denatured) 16 hours >>100 hours Wisk (denatured) 60-70 hours >>100hours

[0267] We also assessed the stability of various enzymes in Cibadetergent #15 at 52° C. The results are depicted in Table VI below:TABLE VI Stability of Subtilisin in Ciba Detergent #15 at 52° C.Activity 15 min T_(1/2) in Initial dilute concentrate Catalyst Activityat 52° C. at 52° C. Alcalase 36 14 ˜15 min Alcalase 33 13 ˜15 min OA 3418 ˜15 min 0.1%, 16 h OA 23 17 ˜30 min 1%, 1 h OA 10 14 ˜30 min 1%, 3 hGA 27 16 ˜40 min 0.05%, 30 min GA 35 15 ˜40 min 0.05%, 10 min

[0268] All of the crosslinked crystals prepared as described in thetable above which had half-lives in detergent concentrate of ˜30 minutesor more also had good solubility profiles.

[0269] In addition, we assessed the stability of various enzymes in Cibadetergent #15 versus Ciba detergent #16 at 40° C. The results aredepicted in the Table VII below: TABLE VII Stability of Subtilisin inCiba Detergent #15 vs. #16 at 40° C. T_(1/2) in #15 T_(1/2) in #16Initial concentrate concentrate Catalyst Activity at 40° C. at 40° C.Alcalase 33 10 h 2.5 h GA 27  7 h ˜10 h 0.05%, 30 min OA 32  9 h   8 h0.1%, 16 h OA 15 12 h  16 h 0.2%, 16 h

[0270] We also assessed the effects of crosslinking time on activity andstability of the resulting crosslinked enzyme crystals. These resultsare summarized in the tables below. In Table VIII, an asterisk indicatesvalues measured by incubating 25 μl in a 2 ml tube and “Xs” denotesuncrosslinked protein crystals.

[0271] In preparing the crosslinked protein crystals described in TablesVIII, IX and X, the protein crystals were crystallized as described inExample 1 and crosslinked with glutaraldehyde as described in Example 2,using the crosslinking times and glutaraldehyde concentrations set forthin that example, or those specified in the tables. TABLE VIIICrosslinking Time vs. Concentration of Glutaraldehyde on Stability ofSubtilisin in Ciba Detergent #16 at 40° C. Activity Activity Stability,Crosslinking abs/mg/min abs/mg/min 18 h GA (%) Time t = 0 t = 18 h % ofXs, t = 0 (Xs) 0 0 33.6 1.1 3.3 (Xs) 0 0 31.7 2.6* 8.2* 0.0189 10.0 28.55.2 16.5 0.0189 10.0 31.3 5.7 17.8 0.0189 39.3 14.1 5.4 17.0 0.0189 39.314.3 5.0 15.8 0.05 5.0 20.7 3.8 12.0 0.05 15.0 16.4 7.0 22.1 0.05 18.619.6 8.7 27.4 0.05 18.6 17.8 9.8 30.9 0.05 60.0 0 13.5 42.6 0.05 60.03.0 14.7 46.4 0.125 3.0 18.3 9.1 28.7 0.125 3.0 15.4 9.0 28.4 0.125 10.07.9 14.9 46.9 0.125 10.0 9.5 14.8 46.6 0.125 10.0 7.9 14.7 46.4 0.12510.0 9.5 13.4 42.1 0.125 10.0 9.4 12.2 38.5 0.125 10.0 8.1 12.2 38.50.125 10.0 8.3 16.0 50.5 0.125 17.0 5.4 14.0 44.2 0.125 17.0 6.4 15.348.3 0.125 39.3 2.1 3.3 10.4 0.125 39.3 1.0 5.8 18.3 0.125 39.3 1.1 4.413.9 0.125 39.3 1.7 4.5 14.3 0.125 39.3 1.6 5.7 18.0 0.125 39.3 0.9 3.310.4 0.125 68.6 1.3 3.1 9.9 0.2 5.0 10.4 12.1 38.2 0.2 15.0 2.5 9.0 28.40.2 18.6 1.8 6.9 21.8 0.2 18.6 0.8 3.1 9.8 0.2 60.0 0.4 1.3 4.1 0.2 60.01.4 1.4 4.4 0.231 10.0 2.7 13.0 41.0 0.231 10.0 4.8 11.7 37.0 0.231 39.30.5 1.1 3.5 Alcalase 28.0 3.1* 9.8* Alcalase 32.9 0.2 1.0

[0272] In Table IX, an asterisk indicates that crystals were crushedduring crosslinking and dash marks indicate that no measurements weretaken at those points. All samples were prepared at 1 ml (27 mg) scale.TABLE IX Activity of Glutaraldehyde Crosslinked Subtilisin in CibaDetergent #16 at 40° C. Cross- linking Activity at 40° C. time(abs/mg/min) GA (%) (min) t = 0 18 h 39 h 63 h 80 h 90 h 6 days (Xs) 0 033.6 1.1 — — — — — 0.0076 60 14.1 3.0 — — — — — 0.02 39 12.0 7.7 — — — —— 0.02 80 6.9 11.5 — — — 0 — 0.02* 80 12.2 26.2 10.7 3 — — — 0.05* 3113.5 26.3 9.8 5.3 — — — 0.05 60 4.9 17.7 7.9 2.4 — — 1.0 0.05* 60 8.727.3 12.5 7.3 — — — 0.05 89 1.3 12.1 — — — 3.7 — 0.08 39 2.2 12.6 — — —5.3 — 0.08 81 0.8 3.9 — —  5.8 — 3.9 0.08* 81 9.8 — — — 10.2 — — 0.125 316.4 9.7 1.7 0.3 — — — 0.125 10 9.4 14.8 9.4 1.9 — — — 0.125 17 6.4 15.311.5 8.5  4.6 — — 0.2 5 10.4 12.7 5.1 0.3 — — — 0.23 10 4.8 11.7 10.08.5  5.2 — — Alcalase 30.5 1.6

[0273] TABLE X Conditions for Larger Scale Crosslinked Enzyme CrystalPreparation—Stability of Glutaraldehyde Crosslinked Subtilisin in CibaDetergent #16 at 40° C. Cross- linking Stability at 40° C. time(abs/mg/min) GA (%) (min) t = 0 18 h (Xs) 0 33.9 — t = 0 16 h 38 h 59 h110 h *0.05 60 23.2 16.8 5.9 1.9 5.6 *0.08 80 14.4 12.2 4.1 2.4 — *0.180 8.0 13.6 5.5 3.1 1.4 *0.125 60 9.3 20.9 13.6 — 2.3 *0.15 80 3.8 11.27.4 5.2 2.8 *0.231 60 5.2 9.9 9.3 6.6 8.3 *1.0 60 1.3 2.5 2.2 1.1 2.4 t= 0 24 h 48 h 72 h 120 h 168 h 264 h §0.25 120 4.8 9.5 7.7 6.7 5.7 4.34.4 §0.20 120 2.6 9.5 8.6 8.7 5.6 — 4.5 §0.15 120 5.2 14.3 9.6 5.6 3.7 —1.2 §0.1-NP/ 5 h/1.5 h 9.6 10.2 6.3 — — 5.7 0.1 GA

[0274] In Table X, an asterisk indicates that crosslinkings were carriedout at a 1-2 g scale, §indicates that crosslinkings were carried out ata 10 g scale on previously crushed crystals and dash marks indicate thatno measurements were taken at those points.

[0275]FIG. 1 graphically depicts the stability of 10 g scalepreparations of crosslinked subtilisin crystals according to thisinvention in Ciba detergent #16 at 40° C. In FIG. 1, “Altus I”represents crystals crosslinked with 0.25% glutaraldehyde for 2 hours;“Altus II” represents crystals crosslinked with 0.20% glutaraldehyde for2 hours; “Altus III” represents crystals crosslinked with 0.15%glutaraldehyde for 2 hours and “Altus IV” represents crystalscrosslinked with 0.1% neopentyl glycol diglycidyl ether for 5 hours,followed by 0.1% glutaraldehyde for 1.5 hours. All the crosslinkedsamples were crushed prior to crosslinking using a Brinkman PolytronHomogenizer, then prepared on a 10 g scale and monitored by theazocasein assay over one week at 40° C.

Example 7 Results of Dissolution Study

[0276] The dissolution study demonstrated whether various crosslinkedenzyme crystals dissolve in concentrate and the extent to which theydissolve upon dilution under conditions of use, for example under washconditions. Representative results of this test are included in thetables below, in which “+” indicates that the sample dissolved, “−”indicates that the sample did not dissolve, “−/+” indicates that thesample dissolved somewhat (1 mg/ml in detergent liquid). In the tables,“GP” denotes crystals crosslinked as described infra for GA crosslinkingusing, instead, ultrapure glutaraldehyde (supplied as an 8% aqueoussolution by the Sigma Chemical Co.) which was not diluted prior toaddition to the protein crystals. TABLE XI Detergent Liquid IncubationStudy - Dissolution Study - Concentrate 14 h at 40° C. Catalyst Ciba #15Ciba #16 Ciba #44 Tide OA − −/+ + − 1%, 16 h OA − − + − 0.5%, 16 h OA− + + − 0.1%, 16 h OA − −/+ + − 0.2%, 16 h GA − −/+ + − 0.5%, 1 h GA − −− − 0.9%, 1 h GA − − + − 0.7%, 1 h EP −/+ + + − 1.0%, 20 min GP − −/+ +− 0.08%, 20 min CLECBL ™ − − − − Crystals + + + − (uncrosslinked)

[0277] TABLE XII Detergent Liquid Incubation Study - Dissolution Study -200 fold Dilution 20 minutes at 52° C. Catalyst Ciba #15 Ciba #16 Ciba#44 Tide OA 1%, 16 h −/+ −/+ + − OA 0.5%, 16 h − + + − OA 0.1%, 16 h− + + + OA 0.2%, 16 h −/+ + + − GA 0.5%, 1 h − −/+ + − GA 0.9%, 1 h− + + − GA 0.7%, 1 h − − + − EP 1.0%,20 min + + + + GP 0.08%,20 min− + + − CLECBL ™ − − − − Crystals (uncrosslinked) + + + +

[0278] As demonstrated in the tables above, crosslinked enzyme crystalsaccording to this invention are essentially insoluble in concentrateddetergent and essentially soluble in diluted detergent under washconditions.

Example 8 Summary of Properties of Crosslinked Enzyme Crystals of thisInvention

[0279] Table XIII below summarizes the overall stability/instability,activity and dissolution properties in Ciba detergent #15 of crosslinkedsubtilisin crystals prepared according to this invention usingdialdehydes. TABLE XIII Solubility Solubility Activity StabilityCrosslinker in Ciba #15 on Dilution (t = 0) at 52° C. Glyoxal lowdissolve at high low 52° C. Succinimaldehyde low dissolve at 17-66% ofND 52° C.; Alcalase partially dissolve at 25° C. Glutaraldehyde very lowdissolve at 1-100% of low 52° C.; Alcalase 52° C. partially to moderatefully 40° C. dissolve at 25° C. Octanedialdehyde very low % dissolve at30-66% of low 52° C.; Alcalase 52° C. partially to moderate fully 40° C.dissolve at 25° C.

[0280] As demonstrated in Table XIII above, the crosslinked enzymecrystals of the present invention are insoluble and, therefore, stableunder storage conditions, while quickly releasing their activity underconditions of use. Advantageously, the crosslinked enzyme crystals ofthis invention exhibit activity similar to their soluble oruncrosslinked crystallized counterparts under conditions of use, whiledisplaying 5-6 fold improved stability, as well as favorable dissolutionproperties.

Example 9 Effect of Change of Chemical Composition on CrosslinkedSubtilisin Crystals

[0281] We crystallized subtilisin as described in Example 1 andcrosslinked the resulting crystals as described in Example 2, using GA1%/1 hour. When 100 μL (2.2 mg) of the resulting crosslinked subtilisincrystals was suspended in 1.5 mL of 33.3% of acetonitrile/phosphatebuffer (0.3 M, pH 7.5), the crystals were completely dissolved after 45minutes at 40° C.

[0282] Using similar conditions, suspending the crosslinked subtilisincrystals in 1.2 mL of 16.7% acetonitrile/buffer, the crystals werecompletely dissolved after 5 hours. Activity (U) in 100% ACN/16.7%ACN/33.3% time buffer Buffer Buffer 0 27.3 27.3 27.3 1.5 h 27.3 — 7.4*3.3 h 27.3 25.5 7.0 h 27.3 24.0**

[0283] Assay: 0.2 mmol (75.8 mg) of TAME in 2.5 mL phosphate buffer wasincubated with each crosslinked subtilisin crystal sample (equal to0.044 mg enzyme crystals) suspension (or solution) at room temperature.One unit hydrolyzed 1.0 μmole of TAME per min. from per mg crosslinkedcrystals.

[0284] The results above illustrate the trigger of addition of organicsolvent to the environment of crosslinked protein crystals of thisinvention.

Example 10 Wash Performance of Detergents Containing CrosslinkedSubtilisin Crystals

[0285] We assessed the activity and storage stability of crosslinkedenzyme crystals of this invention in liquid detergent, using a washingassay designed to test the ability of the detergent to remove stainsfrom a fabric.

Washing Assay

[0286] Preparation of fabric

[0287] Cloth samples of the same size and weight were cut from the samebolt:

[0288] 5 g of soiled test cloth and

[0289] 5 g of cotton ballast with no soil (Ciba No. 1-3005).

[0290] Prior to washing the samples, we measured the light intensity(=lightness) remitted by the soiled fabric samples (as described below).

[0291] Preparation of Detergent Solution

[0292] The sample of liquid detergent to be tested was heated in a flaskfor two hours at 20° C. The sample was then homogenized by vigorousshaking and 0.8 g of the detergent was removed from the flask and addedto 200 ml of tap water (20° C.) in a metallic beaker. The aqueousdetergent solution was stirred for 60 seconds.

[0293] Washing

[0294] A sample of soiled test cloth and a sample of unsoiled ballastwere placed together into the beaker containing the aqueous detergentsolution. The beaker was closed tightly and immediately inserted into apre-heated (40° C.) washing machine (Unitest, manufactured by Hereus,Switzerland). During the washing process, the beaker was rotatedconstantly in a water bath heated to 40° C. As a result, the contents ofthe beaker continuously warmed, up to a temperature of 40° C.

[0295] Exactly 20 minutes after the fabric was placed in the detergentsolution, washing was stopped and the washed fabric was immediatelyremoved from the detergent solution and rinsed for 30 seconds with coldtap water (13-15° C.). The wet fabric was centrifuged and ironed toremove wrinkles and dried at the same time.

[0296] Measurement of Washing Performance

[0297] Each sample of the washed and dried fabric was examined for stainremoval by remission measurements (lightness Y) between 460 and 700 nmusing a Spectraflash 500 (Datacolor). A cut off filter was used toeliminate potential interference by contamination with UV-absorbingmaterials. The lightness value of each test cloth was measured 5×and anaverage calculated.

[0298] With increasing washing performance, the lightness of the fabricincreases. Washing performance is thus defined as a difference inlightness, ΔY:

ΔY=Lightness of fabric after washing−Lightness of fabric before washing

Example 11 Effect of Concentration of Crosslinked Subtilisin Crystals onWashing Performance of Detergents Containing Them

[0299] Washing performance of crosslinked enzyme crystals according tothis invention was examined as a function of their concentration in theliquid detergent, using the materials described below. Test fabric: EMPA(Eidgenossische Materialprufungs und Forschungsanstalt, St. Gallen,Switzerland) #116 soiled with a combination of blood, milk and carbonblack. Liquid detergent: Ciba detergent #16. Enzyme: Crosslinked enzymecrystals; sample Altus IV (as described in Example 6) Uncrosslinkedenzyme (Alcalase).

[0300] Concentration of Enzyme in Liquid Detergent:

[0301] enzyme concentrations were between 0.05 and 0.9 w % (dry matterweight). Table XIV provides further details. TABLE XIV Dry matter LiquidWeight of enzyme weight of Detergent Enzyme suspension (g) enzyme Ciba#16 w % Alcalase Altus IV g g 0.05 0.106 0.0053 10 0.05 0.130 0.0056 100.1 0.207 0.0104 10 0.1 0.240 0.0104 10 0.3 0.599 0.0301 10 0.3 0.6830.0297 10 0.5 0.492 0.0247 5 0.5 0.580 0.0252 5 0.9 0.886 0.0445 5 0.91.046 0.0455 5

[0302] Preparation of Liquid Detergent with Enzyme

[0303] Specific aliquots of the suspension of enzyme crystals (see TableXIV) were added to a flask and centrifuged to separate the crystals fromthe liquid. The liquid was discarded and the crystals were suspended andhomogenized in the liquid detergent (for quantities see Table XIV). Theresulting preparations were used in the washing tests.

[0304] Washing tests to evaluate the performance of the enzyme detergentformulations were carried out as described in the assay above. Theresults of the study are depicted in FIG. 2. The figure demonstratesthat at enzyme concentrations ≧0.1 w %, the washing effect of Cibaliquid detergent #16 formulated with Altus IV exceeds that of theformulation with uncrosslinked Alcalase. The efficacy of bothcrosslinked and uncrosslinked enzymes was reduced at enzymeconcentrations below 0.1 w %.

Example 12 Storage Stability and Washing Performance of DetergentsContaining Crosslinked Subtilisin Crystals

[0305] Detergents formulated with crosslinked and uncrosslinked enzymeswere stored at a constant temperature, in order to examine enzymestability in concentrated liquid detergent. The detergent formulations(150 g each) were prepared by the same procedure as the samples inExample 10.

[0306] Liquid detergent: Ciba detergent #16.

[0307] Enzyme:—Crosslinked enzyme crystals: sample Altus IV (Example 6)

[0308] Uncrosslinked enzyme (Alcalase)

[0309] Enzyme concentration: 0.3 w % (dry matter) in liquid detergent.

[0310] Storage Temperature for Stability Studies:

[0311] All samples were stored at 30° C. for between 0 and 7 days. After7 days, the samples were divided after 7 days into two equal portions,in order to study stability at elevated temperature. One portioncontinued to be stored at 30° C., while the other was stored at 40° C.

[0312] Test fabric: Three different soiled fabrics were used. All ofthem were standard test materials available from EMPA:

[0313] EMPA #112: cocoa soiled fabric

[0314] EMPA #116: blood, milk and carbon black soiled fabric

[0315] EMPA #111: blood soiled fabric.

[0316] Washing Performance on Cocoa Soiled Fabric

[0317] Washing performance of various enzyme formulated liquiddetergents was studied with respect to removal of cocoa stains from acocoa soiled test fabric, using the washing assay described above.Storage stability was determined by assessing washing performanceperiodically during the detergent storage time, thus monitoring theimpact of storage temperature on enzyme performance in the liquiddetergent. In this assay, the effectiveness of the liquid detergentdecreases as enzyme stability degrades. The results of this assay, shownin FIGS. 3 and 4, are discussed below.

[0318] Storage Stability at 30° C.

[0319] As demonstrated in FIG. 3, both Alcalase and Altus IV formulateddetergents exhibited an improved performance after 2 days of storage(compared to initial values). However, as storage time increased, theperformance of the Alcalase formulation decreased continuously overtime, while the Altus IV formulated detergent exhibited no degradation,even after 28 days of storage.

[0320] Storage Stability at 40° C.

[0321] As demonstrated in FIG. 4, when the temperature was raised from30 to 40° C., Alcalase formulated detergent lost activity within 2 days,while the Altus IV formulated detergent degraded slightly, whileremoving the cocoa soil from the test fabric significantly, even after21 days of storage at 40° C.

[0322] Washing Performance on Fabric Soiled by a Combination of Blood,Milk and Carbon Black (EMPA #116 Test Fabric)

[0323] The experimental conditions and detergents were the same (exceptthe stained fabric) as for washing of cocoa stains. The results of thewashing tests are denicted in FIGS. 5 and 6.

[0324] Storage Stability at 30° C.

[0325]FIG. 5 clearly illustrates the decay of washing performance of theAlcalase formulated detergent after 2 days of storage at 30° C. However,liquid detergent containing Altus IV enzyme maintained its originalwashing performance, even after 28 days of storage.

[0326] Storage Stability at 40° C.

[0327] As demonstrated in FIG. 6, when the storage temperature wasraised from 30 to 40° C., Alcalase formulated detergent lost nearly allof its washing performance within 2 days. In contrast, detergentcontaining Altus IV retained its washing power for an additional 14days.

[0328] Washing Performance on Fabric Soiled with Blood

[0329] Washing performance on blood stains was tested with enzymecontaining detergents stored at 30° C. The detergent composition,washing conditions were the same as in washing of cocoa stains. Theresults of the washing test are illustrated in FIG. 7.

[0330] The assays show that the washing effect on blood stain by Ciba#16 liquid detergent formulated with Alcalase was low in comparison todetergent without enzyme. On the other hand, the Altus IV formulationwas more active in washing conditions and more stable in storage.

[0331] Storage Stability at 30° C.

[0332] The washing effect of Alcalase formulated detergent decreasedrapidly with storage time, whereas Altus IV formulated detergentretained almost completely its full capacity after 28 days of storage.

Example 13 Solubility of Crosslinked Subtilisin Crystals at 30° C. and37° C.

[0333] We studied the solubility of various subtilisin crystals, whichhad been crosslinked with glutaraldehyde (GA), octanedialdehyde (OA),neopentyl glycol diglycidyl ether (NP) followed by glutaraldehyde, orDENACOL EX-411 (411) followed by glutaraldehyde.

[0334] In 1.5 ml Eppendorf tubes, samples of uncrosslinked subtilisincrystals and crosslinked subtilisin crystal slurry, equal to 37.5 mg ofenzyme, were microfuged at 5,000 rpm for 5 min and the supernatantliquid was removed. A 1.5 ml aliquot of PBS buffer (0.01 M phosphate,0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) was addedto each sample, bringing the concentration of subtilisin to 25 mg/ml.The samples were transferred to 2 ml glass vials with screw caps andmagnetic stir bars then were incubated at 30° C. or at 37° C. Sampleswere studied for dissolution by periodically removing 50 μl of theslurry, microfuging at 13,000 rpm for 5 mins, removing 20 μl of thealiquot and placing it in 980 μl of deionized water, then measuring UVabsorbance at 280 nm.

[0335] The following samples were studied: Crosslinker CrosslinkerConcentration Crosslinking Time GA  1.0% 1.5 h GA 0.25%   2 h GA  0.2%  2 h GA 0.15%   2 h NP/GA 0.1%/0.1%   5 h/1.5 h 411/GA 0.015%/0.035% 16 h/1 h OA  0.2%  16 h OA  0.1%  16 h OA 0.05%  16 h

[0336] The solubility profiles of the samples, shown in FIGS. 8 and 9,illustrate different rates of dissolution for the crosslinked crystals.

Example 14 Reversible Crosslinkers—Disulfide Crosslinked SubtilisinCrystals

[0337] We prepared subtilisin crystals (30-40 μm average, 27 mg/ml inNa₂SO₄) as previously described for subtilisin crystallization.

[0338] We then crosslinked the crystals using one of the followingcrosslinkers:

[0339] 1) Dimethyl 3, 3′-dithiobispropionimidate.HCl—(DTBP) (Pierce)

[0340] 2) Dithiobis(succinimidylpropionate)—(DSP)(Pierce)

[0341] 3) 3, 3′-Dithiobis (sulfosuccinimidylpropionate)—(DTSSP)(Pierce).

[0342] Crosslinking was carried out in 15 ml neoprene screw cap tubes byplacing 740 μl of subtilisin crystal slurry (20 mg) in 9.26 ml of buffer(25 mM NaCO₃/50 mM NaHCO₃, pH 8.0). One crosslinker was added to eachtube as follows: A) 93 mg DTBP (30 mM) B) 100 mg DTSSP (16 mM) C) 120 mgDSP (30 mM).

[0343] The tubes were tumbled at ambient temperature (24-26° C.) untilall samples were determined to be insoluble in 32 mM NaOH (5 days)-100μl sample in 300 μl NaOH. Uncrosslinked samples were readily soluble in32 mM NaOH at the same concentrations. Crosslinking was stopped by theaddition of 1 ml of 1 M Tris, pH 7.5. The samples were centrifuged at3,000 rpm for 5 minutes, the supernatant removed and replaced by 5 ml of100 mM Tris, pH 7.5. Centrifugation at 3,000 rpm, for 5 min, followed byreplacement of supernatant with 5 ml of 100 mM Tris (pH 7.5) wasrepeated 3×.

Example 15 Dissolution of Disulfide Bond-Containing CrosslinkedSubtilisin Crystals

[0344] A 200 mM solution of cysteine was prepared by dissolving 121 mgcysteine in 5 ml 100 mM Tris (pH 7.5). A 400 μl aliquot of the cysteinesolution was added to 3×750 μl vials. A 400 μl aliquot of 100 mM Tris(pH 7.5) was added to another 3×750 μl vials. Each crosslinked sample(100 μl) was added to one vial containing cysteine and one vial withoutcysteine. All samples were incubated at 37° C. and monitored fordissolution of crosslinked enzyme crystals (direct visual andmicroscopic observation).

[0345] After incubation for 3 hrs at 37° C., the DTBP sample appeared tobe fully soluble in the presence of cysteine and insoluble in itsabsence. The DTSSP sample appeared to be nearly fully soluble in thepresence of cysteine and insoluble in its absence. The DSP sample wasbarely soluble in the presence of cysteine and insoluble in its absence.

Example 16 Crystallization of Candida Rugosa Lipase

[0346] A 5 kg aliquot of Candida rugosa lipase (“CRL”) in powder form(Meito) was mixed with 5 kg celite and dissolved in 102 L distilleddeionized water and the volume brought to 200 L with the deionizedwater. The suspension was mixed in an Air Drive Lightning Mixer for 2hours at room temperature and then filtered through a 0.5 micron filterto remove celite. The mixture was then ultrafiltered and concentrated to14 L (469 g) using a 3K hollow fiber filter membrane cartridge. Solidcalcium acetate was added to a concentration of 5 mM Ca(CH₃COO)₂. The pHwas adjusted to pH 5.5 with concentrated acetic acid as necessary. A 7litre aliquot was crystallized by either addition of 1.75 litres of2-methyl-2,4-pentanediol (“MPD”) or by addition of 3.5 litres of a 30%solution of PEG-8000. The resulting solution was mixed andcrystallization allowed to proceed overnight at ambient temperature forabout 17-20 hrs. The crystal yield was about 70%.

[0347] Recrystallization

[0348] The Candida rugosa lipase crystals were solubilized by theaddition of 50 mM sodium phosphate (pH 5.2). Soluble proteinconcentration of the crystallization solution was adjusted to 20 mg/ml.MPD was added gradually with stirring over a 6-hour period, to a finalconcentration of 25%. The resulting solution was mixed andcrystallization allowed to proceed at ambient temperature for 20 hours.

Example 17 Crystallization of Candida Rugosa Lipase

[0349]Candida rugosa lipase crystals prepared as described in Example16, prior to the solubilization and recrystallization steps, weresolubilized by the addition of 50 mM sodium acetate (pH 6.5). Solubleprotein concentration of the crystallization solution was adjusted to 20mg/ml. MPD was added gradually with stirring over a 6-hour period to afinal concentration of 20%. The resulting solution was mixed andcrystallization allowed to proceed at ambient temperature for 20 hours.

Example 18 Crosslinking of Candida Rugosa Lipase Crystals

[0350]Candida rugosa lipase crystals, prepared as described in Example16, were crosslinked by addition of untreated neat glutaraldehyde(Sigma) by adding 2 ml of 20% glutaraldehyde stepwise in a 40.5 mlvolume over one hour to 8 ml of stirred lipase crystals (25 mg/ml), atambient temperature. The final crosslinker concentration was 4.0%.Crosslinking was allowed to proceed over 24 hours. Crystals wererecovered by low speed centrifugation and washed with 25% MPD in 50 mMsodium phosphate (pH 5.2).

Example 19 Crosslinking of Candida Ruaosa Lipase Crystals

[0351]Candida rugosa lipase crystals, prepared as described in Example16, were crosslinked by addition of untreated neat glutaraldehyde byadding 2 ml of 20% glutaraldehyde gradually over a one hour period.Crystals were crosslinked and processed as described in Example 18.

Example 20 Crosslinking of Candida Rugosa Lipase Crystals

[0352]Candida rugosa lipase crystals, prepared as described in Example16, were crosslinked as described in in Example 19, except that thereaction was allowed to proceed for 24 hours. The crystals were thenprocessed as described in Example 18.

Example 21 Crosslinking of Candida Rugosa Lipase Crystals

[0353]Candida rugosa lipase crystals, prepared as described in Example17, were crosslinked by addition of glutaraldehyde to a finalconcentration of 4.0%. Crosslinking was allowed to proceed for 3 hours.The crystals were processed as described in Example 18.

Example 22 Crosslinking of Candida Rugosa Lipase Crystals

[0354]Candida rugosa lipase crystals, prepared as described in Example17, were crosslinked in neat glutaraldehyde at a concentration of 6.5%for 1 hour. Crosslinking and processing were performed as described inExample 18.

Example 23 Crosslinking of Candida Rugosa Lipase Crystals

[0355]Candida rugosa lipase crystals, prepared as described in Example17, were crosslinked in neat glutaraldehyde at a concentration of 6.0%for 1 hour. Crosslinking and processing were performed as described inExample 18.

Example 24 pH Controlled Solubility of Crosslinked Candida Rugosa LipaseCrystals

[0356] Solubility of various crosslinked Candida rugosa lipase crystalswas studied following an increase in pH from 6.5 to 9.0. The crystalswere incubated at 1 mg/ml in 50 mM sodium phosphate (pH 9) containing25% MPD. Aliquots were removed after 3 hour and 24 hour incubation at25° C. with stirring. Activity and soluble protein concentration weremeasured as described in Example 25. The results are described in thetable below. Time (hr) 3 24 Crosslinked Crystal [Prot.] [Prot.]Preparation Activity (U) (mg/ml) Activity (U) (mg/ml) Example 18 7.50.47 20 1 Example 19 10.8 0.60 11 0.63 Example 20 7.5 0.42 8 0.49

Example 25 pH Solubility of Crosslinked Candida Rugosa Lipase Crystals

[0357] Solubility of various crosslinked Candida rugosa lipase crystalswas studied following an increase in pH from 5.2 to 7.5. The crystalswere incubated at 1 mg/ml in 50 mM sodium phosphate (pH 7.5) containing25% MPD. Aliquots were removed after 3 hour and 24 hour incubation at25° C. with stirring. Insoluble material was removed by filtration (0.25micron). Activity in solution was measured spectrophotometrically bymonitoring the hydrolysis of para nitrophenyl acetate (Fluka) at 400 nm.Substrate concentration was 1 mM. The assay was performed at 25° C. in a1 ml volume of 50 mM sodium acetate (pH 6.5). Soluble proteinconcentration was measured by absorbance at 280 mm. Results arepresented in the table below. Time (hr) 3 24 Crosslinked Crystal [Prot.][Prot.] Preparation Activity (U) (mg/ml) Activity (U) (mg/ml) Example 212.4 0.12 15 0.91 Example 22 10.0 0.63 15 1.0 Example 23 2.5 0.17 11 0.69

Example 26 Crystallization of Human Serum Albumin

[0358] Human serum albumin (“HSA”) was purchased from Sigma ChemicalCompany as a lyophilized powder. We added 10 grams of protein powder toa 75 ml stirred solution of 100 mM phosphate buffer pH 5.5 at 4° C.Final protein concentration was 120 mg/ml (determined from OD₂₈₀,extinction coefficient for serum albumin was assumed to equal 1).Saturated ammonium sulfate solution (767 g/l) prepared in deionizedwater was added to the protein solution to a final concentration of 50%saturation (350 g/l). The crystallization solution was “seeded” with 1ml of albumin crystals (50 mg/ml) in 50% ammonium sulfate (pH 5.5). Seedcrystals were prepared by washing a sample of crystals free ofprecipitate with a solution of 50% saturated ammonium sulfate in 100 mMphosphate buffer (pH 5.5). The seeded crystallization solution wasincubated at 4° C. overnight on a vigorously rotating platform. Crystalrods (20μ) appeared in the solution overnight (16 hr).

Example 27 Crosslinking of Human Serum Albumin Crystals

[0359] We crosslinked human serum albumin crystals, prepared asdescribed in Example 18, at 4° C. in a 10 ml stirred solution ofcrystals and mother liquor containing 50% saturated ammonium sulfate, asdescribed above. The crystals, which were not washed prior tocrosslinking, were crosslinked with glutaraldehyde as supplied by themanufacturer (Sigma). Glutaraldehyde (“GA”) (20%) was added to thestirred crystallization solution in 4 equal volumes (62.5 μl) at 15minute intervals to a final concentration of 0.5% (250 μl GA). Thecrystals were then incubated at 4° C. Aliquots were removed atincubation times 0, 30 min, 60 min and 4 hours incubation. Crosslinkedalbumin crystals were collected by low speed centrifugation and washedrepeatedly with pH 7.5, 100 mM Tris HCl, 4° C. Washing was stopped whenthe crystals could be centrifuged at high speed without aggregation.

Example 28 Crosslinking of Human Serum Albumin Crystals

[0360] We crosslinked human serum albumin crystals as described inExample 27 above, with one modification; glutaraldehyde (20%) was addedto the crystallization solution in 4 equal volumes (131.3 μl) at 15minute intervals to a final concentration of 1% (525 μl GA).

Example 29 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, time: 0 minutes incubation. Dissolution Induced by ElevatedTemperature

[0361] Human serum albumin, crystallized as described in Example 26 andcrosslinked for 0 minutes in 0.5% glutaraldehyde, as described inExample 27, was assayed for solubility by incubating the crystals (20mg/ml) with stirring, in phosphate buffered saline solution (pH 7.5) atroom temperature (“RT”) or at 37° C. Aliquots were removed for assay attimes 0.5, 1, 4 and 24 hours. Insoluble material was removed from thesolution by centrifugation and the soluble protein concentration wasmeasured spectrophotometrically at 280 nm, as indicated in Table XV.TABLE XV Soluble Protein (mg/ml) Time (hr) RT 37° C. 0.5 0.3 1.5 1.0 3 54.0 4 12.5 24.0 17 18.5

Example 30 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, time: 30 minutes incubation. Dissolution Induced by ElevatedTemperature

[0362] Human serum albumin, crystallized as described in Example 26 andcrosslinked in 0.5% glutaraldehyde, as described in Example 27, wasassayed for solubility by incubating the crystals (20 mg/ml) inphosphate buffered saline solution (pH 7.5) at room temperature or at37° C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours.Insoluble material was removed from the solution by centrifugation andthe soluble protein concentration was measured spectrophotometrically at280 nm, as indicated in Table XVI. TABLE XVI Soluble Protein (mg/ml)Time (hr) RT 37° C. 0.5 1.5 4 1.0 3 5.5 4.0 7 10 24.0 13.5 17.5

Example 31 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, time: 60 minutes incubation. Dissolution Induced by ElevatedTemperature

[0363] Human serum albumin, crystallized as described in Example 26 andcrosslinked with 0.5% glutaraldehyde, as described in Example 27, wasassayed for solubility by incubating the crystals (20 mg/ml) inphosphate buffered saline solution (pH 7.5) at room temperature or at37° C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours.Insoluble material was removed from the solution by centrifugation andthe soluble protein concentration was measured spectrophotometrically at280 nm, as indicated in Table XVII. TABLE XVII Soluble Protein (mg/ml)Time (hr) RT 37° C. 0.5 0 0.4 1.0 0 0.6 4.0 0 3 24.0 8 17

Example 32 Solubility of Human Serum Albumin Crystals Crosslinked in0.5% GA, time: 240 minutes incubation. Dissolution Induced by ElevatedTemperature

[0364] Human serum albumin, crystallized as described in Example 26 andcrosslinked with 0.5% glutaraldehyde, as described in Example 27, wasassayed for solubility by incubating the crystals (20 mg/ml) inphosphate buffered saline solution (pH 7.5) at room temperature or at37° C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours.Insoluble material was removed from the solution by centrifugation andthe soluble protein concentration was measured spectrophotometrically at280 nm, as indicated in Table XVIII. TABLE XVIII Soluble Protein (mg/ml)Time (hr) RT 37° C. 0.5 0 0 1.0 0.5 0 4.0 3.5 3 24.0 8.5 14.5

Example 33 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% CA, time: 0 minutes incubation. Dissolution Induced by ElevatedTemperature

[0365] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XIX. TABLE XIX Soluble Protein (mg/ml) Time (hr) RT37° C. 0.5 1 2 1.0 3 7 4.0 10.5 16 24.0 19 18.5

Example 34 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% GA, time: 30 minutes incubation. Dissolution Induced by ElevatedTemperature

[0366] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XX. TABLE XX Soluble Protein (mg/ml) Time (hr) RT 37°C. 0.5 0 0 1.0 0 2 4.0 4.5 7 24.0 8 13

Example 35 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% GA, time: 60 minutes incubation. Dissolution Induced by ElevatedTemperature

[0367] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XXI. TABLE XXI Soluble Protein (mg/ml) Time (hr) RT37° C. 0.5 0 0.5 1.0 0 1.5 4.0 1 4 24.0 9 13.5

Example 35 Solubility of Human Serum Albumin Crystals Crosslinked in1.0% GA, time: 240 minutes incubation. Dissolution Induced by ElevatedTemperature

[0368] Human serum albumin, crystallized as described in Example 26 andcrosslinked as described in Example 27, was assayed for solubility byincubating the crystals (20 mg/ml) in phosphate buffered saline solution(pH 7.5) at room temperature or at 37° C. Aliquots were removed forassay at times 0.5, 1, 4 and 24 hours. Insoluble material was removedfrom the solution by centrifugation and the soluble proteinconcentration was measured spectrophotometrically at 280 nm, asindicated in Table XXII. TABLE XXII Soluble Protein (mg/ml) Time (hr) RT37° C. 0.5 0 0 1.0 0 0 4.0 0 2 24.0 6 10.3

Example 36 Crystallization of Thermolysin

[0369] Thermolysin was purchased from Diawa (Japan) as a lyophilizedpowder. Fifteen grams of protein powder were added to a 100 ml stirredsolution of 10 mM calcium acetate (pH 11) at ambient temperature. The pHwas maintained at 11 by addition of 2 N NaOH, until the thermolysin wascompletely solubilized. The pH was then adjusted to pH 7.5 by additionof 2 N acetic acid. Crystallization was allowed to proceed overnight at4° C. Final protein concentration was 40 mg/ml (determined from OD₂₈₀,extinction coefficient for thermolysin was assumed to equal 1.8).Crystals were recovered by centrifugation and recrystallized to obtain amore uniform crystal size. Recrystallization was performed in a mannernearly identical to that described for the initial crystallization.Crystals (40 mg/ml protein) were dissolved by addition of base at roomtemperature. The pH of the crystallization solution was adjusted to 6.5and crystallization was permitted to proceed at ambient temperature.Crystal rods (50 u) appeared in the solution overnight (16 hr).

Example 37 Crosslinking of Thermolysin Crystals

[0370] Thermolysin crystals, prepared as described in Example 36, weresuspended (50 mg/ml) in a 50 mM solution of sodium acetate (pH 6.5).Crystals were crosslinked with glutaraldehyde as supplied by themanufacturer (Sigma). Ten milliliters of glutaraldehyde (10%) were addedgradually over a 1 hour period with stirring to a 10 ml suspension ofcrystals. After all of the glutaraldehyde was added, the crystallizationsolution incubated at ambient temperature. Aliquots were removed atincubation times 0.5, 1 and 3 hr. Crosslinked crystals were collected bylow speed centrifugation and washed exhaustively with pH 7.5 50 mM TrisHCl, containing 10 mM calcium acetate.

Example 38 Solubility of Thermolysin Crystals Crosslinked for 0.5 hr.Dissolution Induced by Removal of Calcium Ions by EDTA

[0371] Thermolysin, crystallized as described in Example 36 andcrosslinked for 0.5 hr as described in Example 37, was assayed forsolubility by incubating the crystals (1 mg/ml) with stirring, in 10 mMTris HCl (pH 7.2) containing 1 mM EDTA (Sigma) 40° C. One ml aliquotswere removed for assay at times 0.5, 3 and 24 hours. Insoluble crystalswere removed from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 μmole of substrate in one minute at pH 7.2, 40°C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXIII. TABLE XXIII Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 80 30 3.0 103 97 24.0 100 91

Example 39 Solubility of Thermolysin Crystals Crosslinked for 1 hr.Dissolution Induced by Removal of Calcium Ions by EDTA

[0372] Thermolysin, crystallized as described in Example 36 andcrosslinked for 1 hr as described in Example 37, was assayed forsolubility by incubating the crystals (1 mg/ml) with stirring, in 10 mMTris HCl (pH 7.2) containing 1 mM EDTA 40° C. One ml aliquots wereremoved for assay at times 0.5, 3 and 24 hours. Insoluble crystals wereremoved from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 μmole of substrate in one minute at pH 7.2, 40°C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXIV. TABLE XXIV Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 7 11 3.0 24 29 24.0 104 87

Example 40 Solubility of Thermolysin Crystals Crosslinked for 3 hr.Dissolution Induced by Removal of Calcium Ions by EDTA

[0373] Thermolysin, crystallized as described in Example 36 andcrosslinked for 3 hr as described in Example 37, was assayed forsolubility by incubating the crystals (1 mg/ml) with stirring, in 10 mMTris HCl (pH 7.2) containing 1 mM EDTA 40° C. One ml aliquots wereremoved for assay at times 0.5, 3 and 24 hours. Insoluble crystals wereremoved from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 μmole of substrate in one minute at pH 7.2, 40°C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXV. TABLE XXV Soluble Protein Activity Time (hr) (%of Max) (% of Max) 0.5 2 0 3.0 2 0 24.0 100 73

Example 41 Solubility of Thermolysin Crystals Crosslinked for 3 hr.Dissolution Induced by Removal of Calcium Ions by Dilution

[0374] Thermolysin crystals, prepared as described in Example 36 andcrosslinked for 3 hr as described in Example 37, were washed free ofcalcium containing buffer and assayed for solubility by incubating thecrystals (1 mg/ml) with stirring in deionized water. One ml aliquotswere removed for assay at times 0.5, 3 and 24 hours. Insoluble crystalswere removed from the solution by filtration. One ml of 500 mM calciumacetate (pH 7.2) was added to each aliquot. Soluble proteinconcentration was measured spectrophotometrically at 280 nm. Enzymaticactivity was measured spectrophotometrically by monitoring thehydrolysis of a dipeptide substrate, FAGLA (Feder). Substrateconcentration was 1.67 mM. One unit is defined as the amount of enzymerequired to hydrolyze 1 μmole of substrate in one minute at (pH 7.2),40° C. The activity of soluble thermolysin was 27 U/mg protein. Data ispresented in Table XXVI. TABLE XXVI Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 0 0 3.0 0 7 24.0 111 81

Example 42 Crystallization of Glucose Isomerase

[0375] Glucose isomerase (“GA”) was supplied by Cultor (Finland) as acrystal slurry. The enzyme was recrystallized by solubilizing a 50 mlvolume of the crystal slurry at 50° C. with stirring for 15 minutes. Thesolution was clarified by filtration and allowed to cool slowly at roomtemperature. Fifty micron crystals appeared within 5 hours. Crystalswere recovered by low speed centrifugation and washed with 166 mMmagnesium sulfate.

Example 43 Crosslinking of Glucose Isomerase Crystals

[0376] Five hundred milligrams of glucose isomerase crystals, preparedas described in Example 42, were suspended in a 50 ml solution of 166 mMmagnesium sulfate. The crystals were crosslinked with glutaraldehyde assupplied by the manufacturer (Sigma). Five milliliters of glutaraldehyde(10%) were added gradually over a 1 hour period with stirring to the 50ml suspension. After all of the glutaraldehyde was added, thecrystallization solution incubated at ambient temperature. Aliquots wereremoved at incubation times 1, 3 and 24 hr. Crosslinked crystals werecollected by low speed centrifugation and washed exhaustively with 50 mMTris HCl (pH 7.0).

Example 44 Solubility of Glucose Isomerase Crystals Crosslinked for 1hr. Dissolution Induced by Removal of Calcium Ions by Dilution at 50° C.

[0377] Glucose isomerase crystals, prepared as described in Example 42and crosslinked for 1 hr as described in Example 43, assayed forsolubility by incubating the crystals (1 mg/ml) with stirring indeionized water. One ml aliquots were removed for assay at times 1, 3and 24 hours. Soluble protein concentration was measuredspectrophotometrically at 280 nm (OD280) (extinction coefficient for GIwas assumed to equal 1). Enzymatic activity was measuredspectrophotometrically by monitoring the conversion of fructose toglucose.

[0378] Glucose concentration was quantitated spectrophotometricallyusing a coupled enzyme assay containing hexokinase andglucose-6-phosphate dehydrogenase. The dehydrogenase uses NADP as acofactor and the amount of NADPH formed in the reaction isstoichiometric with the concentration of substrate (glucose). The assaywas purchased as a kit from Boehringer Mannheim and was used accordingto the manufacturer's instructions. One unit is defined as the amount ofenzyme required to convert 1 μmole fructose to glucose in one minute atpH 7.0, 60° C. The activity of soluble glucose isomerase was 51 U/mgprotein. Data is presented in Table XXVII. TABLE XXVII Soluble ProteinActivity Time (hr) (% of Max) (% of Max) 0.5 8 0 3.0 52 31 24.0 100 57

Example 45 Solubility of Glucose Isomerase Crystals Crosslinked for 3hr. Dissolution Induced by Removal of Calcium Ions by Dilution at 50° C.

[0379] Glucose isomerase crystals, prepared as described in Example 42and crosslinked for 3 hr as described in Example 43, were assayed forsolubility by incubating the crystals (1 mg/ml) with stirring indeionized water. One ml aliquots were removed for assay at times 1, 3and 24 hours. Soluble protein concentration was measuredspectrophotometrically at 280 nm (OD280) (extinction coefficient for GIwas assumed to equal 1). Enzymatic activity was measuredspectrophotometrically by monitoring the conversion of fructose toglucose.

[0380] Glucose concentration was quantitated spectrophotometricallyusing the coupled enzyme assay containing hexokinase andglucose-6-phosphate dehydrogenase, as described in Example 44. Data ispresented in Table XXVIII. TABLE XXVIII Soluble Protein Activity Time(hr) (% of Max) (% of Max) 0.5 2 0 3.0 10 6.5 24.0 86 43

Example 46 Solubility of Glucose Isomerase Crystals Crosslinked for 24hr. Dissolution Induced by Removal of Calcium Ions by Dilution at 50° C.

[0381] Glucose isomerase crystals, prepared as described in Example 42and crosslinked for 1 hr as described in Example 43, were assayed forsolubility by incubating the crystals (1 mg/ml) with stirring indeionized water. One ml aliquots were removed for assay at times 1, 3and 24 hours. Soluble protein concentration was measuredspectrophotometrically at 280 nm (OD280) (extinction coefficient forglucose isomerase was assumed to equal 1). Enzymatic activity wasmeasured spectrophotometrically by monitoring the conversion of fructoseto glucose.

[0382] Glucose concentration was quantitated spectrophotometricallyusing the coupled enzyme assay containing hexokinase andglucose-6-phosphate dehydrogenase, as described in Example 44. Data ispresented in Table XXIX. TABLE XXIX Soluble Protein Activity Time (hr)(% of Max) (% of Max) 0.5 2 0 3.0 24 5 24.0 83 61

Example 47 Preparation of Tablets Containing Crosslinked ProteinCrystals According to this Invention

[0383] Tablets containing crosslinked protein crystals according to thisinvention may be prepared as follows. A suspension of crosslinkedprotein crystals is placed in 0.1 M sodium acetate, 20 mM calciumchloride and buffer (pH 7) and dried at 35° C. The resulting driedmaterial may be mixed with sorbitol 50:50 by weight and granulated withEudragit NE 30D (a neutral copolymer based on ethyl- and methylacrylate)or Eudagit RL 30D (an ammonio-methacrylate copolymer). The granules aredried (for example, for 16 hours at 40° C.) and compressed to roundtablets of about 5 mm diameter and weight of about 125 mg. The contentof crosslinked protein crystals in such solid preparations is about 45%by weight. If the above-described preparation is made without usingsorbitol, the resulting tablets contain about 63% by weight crosslinkedprotein crystals.

[0384] When introduced into water or aqueous buffer (such as theabove-described acetate buffer) all the tablets disintegrate in a matterof 10 minutes under mild shaking at room temperature) producingparticles less than 100 μm in size, the majority in the range of 4-10μm. Microscopic examination reveals polymer-free singular proteincrystals, as the predominant species. The slurry obtained by disruptingthe tablets is assayed titrimetrically using hydrolysis of N(α)-p-tosylL-arginine methyl ester (TAME) at 25° C. (pH 8). Activity correspondingto between about 50% and 80% of activity of an equal amount ofcrosslinked protein crystals (counting the indicated weight of thecrosslinked crystals, rather than of the whole tablets) results.

Example 48 Crystallization of Candida Rugosa Lipase

[0385]Candida rugosa lipase was prepared as described in Example 16.After the addition of solid calcium acetate to 5 mM, however, the pH wasadjusted to 4.8 instead of pH 5.5 with concentrated acetic acid asnecessary. Next, a seven liter aliquot was crystallized by the additionof 1.4 liters of MPD. The resulting solution was mixed andcrystallization was allowed to proceed for 72 hours at 4° C.

[0386] In the following examples, unless otherwise indicated, lipasecrystals were prepared according to this example.

Example 49 Crosslinking of Lipase Crystals

[0387] Lipase crystals were crosslinked using the followingcrosslinkers: dimethyl 3, 3′-dithiobispropionimidate.HCl (DTBP);dithiobis (succinimidylpropionate) (DSP); bismaleimidohexane (BMH);bis[Sulfosuccinimidyl]suberate (BS); 1,5-difluoro-2,4-dinitrobenzene(DFDNB); dimethylsuberimidate.2HCl (DMS); disuccinimidyl glutarate(DSG); disulfosuccinimidyl tartarate (Sulfo-DST);1-ethyl-3-[3-dimethylaminoproplyl]carbodiimide hydrochloride (EDC);ethylene glycolbis [sulfosuccinimidylsuccinate] (Sulfo-EGS);N-[γ-maleimidobutyryloxy]succinimide ester (GMBS);N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB);sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate(Sulfo-LC-SMPT); bis-[β-(4-azidosalicylamido) ethyl]disulfide (BASED);NHS-PEG-Vinylsulfone (NHS-PEG-VS); and glutaraldehyde (GA).

[0388] Dimethyl 3,3′-dithiobispropionimidate-HCl(DTBP) Crosslinking

[0389] A dimethyl 3,3′-dithiobispropionimidate-HCl (DTBP) solution wasprepared by dissolving 27.9 mg of DTBP in 60 μl of water. Next, 40 μl ofthis solution was added to 21 mg of lipase crystals in 1.5 ml of 10 mMHEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD.The crosslinking reaction was carried out at ambient temperature for 24hours with tumbling. After 24 hours, the slurry was centrifuged at 3000rpm for 5 minutes and the supernatant was discarded. The pellet was thensuspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calciumchloride and 20% MPD. An additional amount (20 μl) of DTBP solution wasadded and crosslinking was continued for another 24 hours. Thecrosslinking reaction was terminated by washing off the excess reagentwith 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calciumchloride and 20% MPD (five washes with 1 ml of buffer).

[0390] Dithiobis(succinimidylpropionate) (DSP) Crosslinking

[0391] A dithiobis (succinimidylpropionate) (DSP) solution was preparedby dissolving 36 mg of DSP in 60 μl of dimethyl sulfoxide (DMSO). Next,40 μl of this solution was added to 21 mg of lipase crystals in 1.5 mlof 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and20% MPD. The crosslinking reaction was carried out at ambienttemperature for 24 hours with tumbling. After 24 hours, the slurry wascentrifuged at 3000 rpm for 5 minutes and the supernatant was discarded.The pellet was then suspended in 10 mM HEPES buffer, pH 7.5 andcontaining 10 mM calcium chloride and 20% MPD. An additional amount (20μl) of DSP solution was added and crosslinking was continued for another24 hours. The crosslinking reaction was terminated by washing off excessreagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mMcalcium chloride and 20% MPD (five washes with 1 ml of buffer).

[0392] Bis Maleimidohexane (BMH) Crosslinking

[0393] A bis maleimidohexane (BMH) solution was prepared by dissolving12 mg of BMH in 40 μl of dimethyl sulfoxide (DMSO). Next, 40 μl of thissolution was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPESbuffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. Thecrosslinking reaction was carried out at ambient temperature for 24hours with tumbling. After 24 hours, the slurry was centrifuged at 3000rpm for 5 minutes and the supernatant was discarded. The pellet was thensuspended in 10 mM HEPES buffer, pH 7.5 containing and 10 mM calciumchloride and 20% MPD. An additional amount (20 μl) of BMH solution wasadded and crosslinking was continued for another 24 hours. Thecrosslinking reaction was terminated by washing off the excess reagentwith 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calciumchloride and 20? MPD (five washes with 1 ml of buffer).

[0394] Bis [Sulfosuccinimidyl]suberate (BS) Crosslinking

[0395] A bis [sulfosuccinimidyl]suberate (BS) solution was prepared bydissolving 29 mg of BS in 50 μl of water. Next, 40 μl of this solutionwas added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer,pH 8.5 and containing 10 mM calcium chloride and 20% MPD. Thecrosslinking reaction was carried out at ambient temperature for 24hours with tumbling. After 24 hours, the slurry was centrifuged at 3000rpm for 5 minutes and the supernatant was discarded. The pellet was thensuspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calciumchloride and 20% MPD. An additional amount (20 μl) of BS solution wasadded and crosslinking was continued for another 24 hours. Thecrosslinking reaction was terminated by washing off the excess reagentwith 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calciumchloride and 20% MPD (five washes with 1 ml of buffer).

[0396] 1,5-Difluoro-2,4-dinitrobenzene (DFDNB) Crosslinking

[0397] A 1,5-Difluoro-2,4-dinitrobenzene (DFDNB) solution was preparedby dissolving 10 mg of DFDNB in 40 μl of acetone. Next, 40 μl of thissolution was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPESbuffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. Thecrosslinking reaction was carried out at ambient temperature for 24hours with tumbling. After 24 hours, the slurry was centrifuged at 3000rpm for 5 minutes and the supernatant was discarded. The pellet was thensuspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calciumchloride and 20% MPD. An additional amount (20 μl) of DFDNB solution wasadded and crosslinking was continued for another 24 hours. Thecrosslinking reaction was terminated by washing off the excess reagentwith 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calciumchloride and 20% MPD (five washes with 1 ml of buffer).

[0398] Dimethylsuberimidate.2HCl (DMS) Crosslinking

[0399] A dimethylsuberimidate.2HCl (DMS) solution was prepared bydissolving 33 mg of DMS in 40 μl of dimethyl sulfoxide (DMSO). Next, 40μl of this solution was added to 21 mg of lipase crystals in 1.5 ml of10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20%MPD. The crosslinking reaction was carried out at ambient temperaturefor 24 hours with tumbling. After 24 hours, the slurry was centrifugedat 3000 rpm for 5 minutes and the supernatant was discarded. The pelletwas then suspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mMcalcium chloride and 20% MPD. An additional amount (20 μl) of DMSsolution was added and crosslinking was continued for another 24 hours.The crosslinking reaction was terminated by washing off the excessreagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mMcalcium chloride and 20% MPD (five washes with 1 ml of buffer).

[0400] Disuccinimidyl Glutarate (DSG) Crosslinking

[0401] A disuccinimidyl glutarate (DSG) solution was prepared bydissolving 17 mg of DSG in 50 μl of dimethyl sulfoxide (DMSO). Next, 40μl of this solution was added to 21 mg of lipase crystals in 1.5 ml of10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20%MPD. The crosslinking reaction was carried out at ambient temperaturefor 24 hours with tumbling. After 24 hours, the slurry was centrifugedat 3000 rpm for 5 minutes and the supernatant was discarded. The pelletwas then suspended in 10 mM HEPES buffer, pH 7.5 containing 10 mMcalcium chloride and 20% MPD. An additional amount of DSG solution (20μl) was added and crosslinking was continued for another 24 hours. Thecrosslinking reaction was terminated by washing off the excess reagentwith 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calciumchloride and 20% MPD (five washes with 1 ml of buffer).

[0402] Disulfosuccinimidyl tartarate (Sulfo-DST) Crosslinking

[0403] A disulfosuccinimidyl tartarate (Sulfo-DST) solution was preparedby dissolving 27 mg of Sulfo-DST in 50 up of water. Next, 40 μl of thissolution was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPESbuffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. Thecrosslinking reaction was carried out at ambient temperature for 24hours with tumbling. After 24 hours, the slurry was centrifuged at 3000rpm for 5 minutes and the supernatant was discarded. The pellet was thensuspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calciumchloride and 20% MPD. An additional amount (20 μl) of Sulfo-DST solutionwas added and crosslinking was continued for another 24 hours. Thecrosslinking reaction was terminated by washing off the excess reagentwith 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calciumchloride and 20% MPD (five washes with 1 ml of buffer).

[0404] 1-Ethyl-3-[3-Dimethylaminopropyl] Carbodiimide Hydrochloride(EDC) Crosslinking

[0405] A 1-Ethyl-3-[3-Dimethylaminopropyl] carbodiimide hydrochloride(EDC) solution was prepared by dissolving 10 mg of EDC in 1 ml of water.Next, 200 μl of this solution and 5 mg of solid Sulfo-NHS was added to21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 andcontaining 10 mM calcium chloride and 20% MPD. The crosslinking reactionwas carried out at ambient temperature for 24 hours with tumbling. After24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and thesupernatant was discarded. The pellet was then suspended in 50 mM MESbuffer, pH 6 and containing 10 mM calcium chloride and 20% MPD. Anadditional amount of an EDC+Sulfo-NHS solution (200 μl+5 mg Sulfo-NHS)was added and crosslinking was continued for another 24 hours. Thecrosslinking reaction was terminated by washing off the excess reagentwith 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calciumchloride and 20% MPD (five washes with 1 ml of buffer).

[0406] Ethylene glycolbis[sulfosuccinimidylsuccinate] (Sulfo-EGS)Crosslinking

[0407] An ethylene glycolbis [sulfosuccinimidyl succinate] (Sulfo-EGS)solution was prepared by dissolving 33 mg of Sulfo-EGS in 40 μl water.Next, 40 μl of this solution was added to 21 mg of lipase crystals in1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calciumchloride and 20% MPD. The crosslinking reaction was carried out atambient temperature for 24 hours with tumbling. After 24 hours, theslurry was centrifuged at 3000 rpm for 5 minutes and the supernatant wasdiscarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5and containing 10 mM calcium chloride and 20% MPD. An additional amount(20 μl) of Sulfo-EGS solution was added and crosslinking was continuedfor another 24 hours. The crosslinking reaction was terminated bywashing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8and containing 10 mM calcium chloride and 20% MPD (five washes with 1 mlof buffer).

[0408] N-[γ-maleimidobutyryloxy]succinimide Ester (GMBS) Crosslinking

[0409] An N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) solution wasprepared by dissolving 23 mg of GMBS in 50 μl of dimethyl sulfoxide(DMSO). Next, 40 μl of this solution was added to 21 mg of lipasecrystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mMcalcium chloride and 20% MPD. The crosslinking reaction was carried outat ambient temperature for 24 hours with tumbling. After 24 hours, theslurry was centrifuged at 3000 rpm for 5 minutes and the supernatant wasdiscarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5and containing 10 mM calcium chloride and 20% MPD. An additional amount(20 μl) of GMBS solution was added and crosslinking was continued foranother 24 hours. The crosslinking was terminated by washing off theexcess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer).

[0410] N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB)Crosslinking

[0411] An N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB)solution was prepared by dissolving 5 mg of Sulfo-HSAB in 40 μl ofwater. Next, 40 μl of this solution was added to 21 mg of lipasecrystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mMcalcium chloride and 20% MPD. The crosslinking reaction was carried outat ambient temperature for 24 hours with tumbling. After 24 hours, theslurry was centrifuged at 3000 rpm for 5 minutes and the supernatant wasdiscarded. The pellet was then suspended in 10 mM HEPES buffer, pH 8.5and containing 10 mM calcium chloride and 20% MPD. A second crosslinkingreaction was carried out at ambient temperature for 10 minutes withshaking and using a 254 nm UV light. The UV lamp was kept 2.5 cm awayfrom the sample. After 10 minutes, the slurry was centrifuged at 3000rpm for 5 minutes and the supernatant was discarded. The crosslinkingreaction was terminated by washing off the excess reagent with 10 mMsodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and20% MPD (five washes with 1 ml of buffer).

[0412] Sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (Sulfo-LC-SMPT) Crosslinking

[0413] A sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (Sulfo-LC-SMPT) solution was prepared by dissolving 12 mg ofSulfo-LC-SMPT in 60 μl of water. Next, 40 μl of this solution was addedto 21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 andcontaining 10 mM calcium chloride and 20% MPD. The crosslinking reactionwas carried out at ambient temperature for 24 hours with tumbling. After24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and thesupernatant was discarded. The pellet was then suspended in 10 mM HEPESbuffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. Anadditional amount (20 μl) of Sulfo-LC-SMPT solution was added andcrosslinking was continued for another 24 hours. The crosslinkingreaction was terminated by washing off the excess reagent with 10 mMsodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and20% MPD (five washes with 1 ml of buffer).

[0414] Bis-[β-(4-azidosalicylamido) ethyl] Disulfide (BASED)Crosslinking

[0415] A bis [β-(4azidosalicylamido) ethyl]disulfide (BASED) solutionwas prepared by dissolving 3 mg of BASED in 40 μl of dimethyl sulfoxide(DMSO). Next, 40 μl of this solution was added to 21 mg of lipasecrystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mMcalcium chloride and 20% MPD. The crosslinking reaction was carried outat ambient temperature for 30 minutes with shaking under a 365 nm UVlight. The UV lamp was shown on the sample from 2.5 cm away. After 30minutes, the slurry was centrifuged at 3000 rpm for 5 minutes and thesupernatant was discarded. The crosslinking reaction was terminated bywashing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8and containing 10 mM calcium chloride and 20% MPD (five washes with 1 mlof buffer).

[0416] NHS-PEG-Vinylsulfone (NHS-PEG-VS) Crosslinking

[0417] An NHS-PEG-Vinylsulfone (NHS-PEG-VS) solution was prepared bydissolving 20 mg of NHS-PEG-VS in 50 μl in water. Next, 50 μl of thissolution was added to 34 mg of lipase crystals prepared as in Example 16in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calciumchloride and 20% MPD. The crosslinking reaction was carried out atambient temperature for 24 hours with tumbling. After 24 hours, theslurry was centrifuged at 3000 rpm for 5 minutes and the supernatant wasdiscarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5and containing 10 mM calcium chloride and 20% MPD. An additional amount(25 μl) of GMBS solution was added and crosslinking was continued foranother 24 hours. The crosslinking was terminated by washing off theexcess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer).

[0418] Glutaraldehyde (GA) Crosslinking

[0419]Candida rugosa lipase crystals were prepared as described inExample 16. The crosslinking reaction was initiated by the addition ofuntreated, neat glutaraldehyde (Sigma) to the crystal solution toachieve a final crosslinker concentration of 0.3% or 0.5%. Thecrosslinking reaction was then allowed to proceed for 1 hour. Thecrosslinked crystals were recovered by low speed centrifugation andwashed with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mMcalcium acetate and 20% MPD.

Example 50 pH Solubility of Crosslinked Candida Rugosa Lipase Crystalsat 37° C.

[0420] The solubility of Candida rugosa lipase crystals crosslinked asdescribed in Example 49 was evaluated. Crystals crosslinked with thefollowing crosslinkers were included dimethyl 3,3′-dithiobispropionimidate.HCl (DTBP), dithiobis(succinimidylpropionate) (DSP), bismaleimidohexane (BMH),bis[Sulfosuccinimidyl] suberate (BS), 1,5-difluoro-2,4-dinitrobenzene(DFDNB), dimethylsuberimidate.2HCl (DMS), disuccinimidyl glutarate(DSG), disulfosuccinimidyl tartarate (Sulfo-DST),1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),ethylene glycolbis [sulfosuccinimidylsuccinate] (Sulfo-EGS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate(Sulfo-LC-SMPT), bis-[β-(4-azidosalicylamido) ethyl]disulfide (BASED)and glutaraldehyde (GA).

[0421] Samples of uncrosslinked lipase crystals prepared as in Example16 and crosslinked lipase crystal slurry equal to 2.8 mg of crystallineenzyme were dispensed into 1.5 ml Eppendorf tubes and microfuged at 3000rpm for 5 minutes. The supernatant liquid was removed and the solubilityof the resulting crystals was evaluated at pH 7.4 and pH 2.0.

[0422] For pH 7.4, a 200 μl aliquot of a 0.01 M phosphate buffer with0.0027 M potassium chloride and 0.137 M sodium chloride at pH 7.4 (PBS)was added to each sample. Under these conditions the concentration oflipase was 14 mg/ml. Next, the samples were incubated at 37° C. for 24hours.

[0423] For the pH 2.0 dissolution measurement, the crosslinked crystalsamples were initially equilibrated in a 10 mM glycine.HCl buffer. Theequilibration buffer was prepared by mixing 80% of 10 mM glycine-HClbuffer with 10 mM calcium chloride at pH 2.0, and 20% of MPD, for afinal pH of 4.8. Equilibration was carried out overnight at 25° C. withtumbling. The equilibrated samples were then microfuged at 300 rpm for 5minutes and the supernatant liquids discarded. The samples werecentrifuged at 3000 rpm for 5 minutes and the pellet was suspended in200 μl of the Glycine.HCl buffer, pH 2.0. Under these conditions, theconcentration of lipase was 14 mg/ml. Finally, the samples wereincubated at 37° C. for 5 hours.

[0424] The amount of soluble protein in the supernatant after the 5 hourincubation at pH 2.0 or the 24 hour incubation at pH 7.4 was measuredusing the Bio-Rad Micro-protein assay. After the incubation period, thesamples were centrifuged at 14,000 rpm for 5 minutes. Next, thesupernatant was filtered through 0.22 μm cellulose acetate filter (SigmaChemical Co.). The soluble protein concentration was then measured bydiluting 2 μl of the filtered supernatant into 798 μl of deionizedwater. Next, 200 μl of Bio-Rad Protein assay reagent was added to thissample and incubated at ambient temperature for 5 minutes. Theabsorbance was measured at 595 nm wavelength and compared to a proteinstandard curve of 0-20 μg bovine serum albumin from Pierce.

[0425] Different triggers alone or in combination determine the kineticsof protein release. The following samples were studied and the resultsare described in the Table XXX below. TABLE XXX Agitation Agitation andAcid Agitation (PBS) pH 2.0 and Pronase Trigger^(→) Protein in filtratein mg Crosslinker (24 hours) (5 hours) (2 hours) DTBP 0.82 0.73 0.81 DSP0.74 0.84 0.68 BMH 0.86 1.74 0.53 DSG 0.94 0.79 0.68 SULFO-DST 1.13 0.850.75 DFDNB 0 0.01 0.06 BASED 0.89 0.99 0.63 GMBS 0.9 0.86 0.83 BS 0.02 00.03 SULFO-HSAB 0.24 0.22 0.22 SULFO-EGS 1.29 0.46 0.64 SULFO-SMPT 0.40.09 0.06 EDC 0.07 0.16 0.02 DMS 1.09 0.83 0.81 GA (0.3%) 0.98 0.97 0.13GA (0.5%) 0.5 0.09 0.02 Soluble 2.8 2.8 2.8

Example 51 Lipase Activity Using Olive Oil as

[0426] The activity of various crosslinked enzyme crystals was assessedby measuring the hydrolysis of a substrate containing olive oil. Duringthe reaction, the pH was held constant by titrating with 0.05 M NaOH.The reaction was followed with a pH-Stat (Titralab™) electrode fromRadiometer, controlled by a VIT90 Video Titrator and a ABU91Autoburette.

[0427] Procedure:

[0428] First, 20 ml of olive oil emulsion was added to the reactionvessel and the reaction mixture was equilibrated to 37° C. withstirring. Next, the pH was adjusted to 7.7 using 0.05 M NaOH and analiquot (34 mg) of crosslinked crystals was added. The pH of the mixturewas maintained at 7.7 by titrating with NaOH. The volume of baseconsumption vs. time (ml/min) was recorded and plotted. The slope of theinitial linear portion of the curve was used to determine the initialreaction rate.

[0429] a) Temperature of reaction: assay vessel was equilibrated andmaintained in 37° C. water bath during course of reaction.

[0430] b) Calculation: Initial rate=base consumption=ml/min (NaOH)×min.

[0431] c) Specific activity (μ moles/min/mG protein)=initialrate×1000×concentration of the titrant/the amount of enzyme (mg).

[0432] d) Blank: Without enzyme—Buffer used in the place of enzyme inthe reaction mixture.

[0433] Reagents:

[0434] a) 3.0 M NaCl (solution A): 34.8 grams of NaCl was added to 150ml of distilled water and stired. Final volume was made up to 200 ml.

[0435] b) 75 mM CaCl₂.2H₂O (solution B): 220 mg of CaCl₂.2H₂O was addedto 150 ml of distilled water with stirring. Once the CaCI₂.2H₂Odissolved, the volume was made up to 200 ml.

[0436] c) Mix: Solution A (40 ml) was added to solution B (20 ml) andH₂O (100 ml).

[0437] d) 0.5% Albumin: 500 mg of albumin/100 ml distilled water, wasprepared by dissolving 1 gm of bovine serum albumin Fraction V (Sigma)in water with gentle stirring (avoid forming foam). After dissolving,the volume was made up to 200 ml.

[0438] e) An olive oil emulsion was prepared by first dissolving 16.5 gmof gum arabic (Sigma) in 130 ml of reagent grade water. Once the gum haddissolved, the volume was increased to 180 ml with distilled water andthe solution was filtered through cotton. Next, 20 ml of olive oil(Sigma) was added and an emulsion was generated by mixing in a QuickPrep mixer for 3 minutes.

[0439] f) Substrate: 50 ml of olive oil emulsion was added to 40 ml ofMix (c) and 10 ml of 0.5% albumin.

Example 52 Activity of Crosslinked Candida Rugosa Lipase Crystals

[0440] The activity of Candida rugosa lipase crystals crosslinked asdescribed in Example 51 was assayed using olive oil as substrate (TableXXXI). Crystals crosslinked with the following crosslinkers wereassayed: dithiobis (succinimidylpropionate) (DSP); bismaleimidohexane(BMH); NHS-PEG-Vinylsulfone (NHS-PEG-VS); disuccinimidyl glutarate(DSG); 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride(EDC); sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT); and glutaraldehyde (GA). TABLE XXXICrosslinked Crystal Volume Specific Activity Preparation (34 mg) addedμl units/mg DSP 40 921 BMH 40 1623 NHS-PEG-VS 50 948 DSG 20 933 DSG 40687 DSG 80 419 EDC 50 1420 EDC 100 1271 EDC 150 686 sulfo-LC-SMPT 205756 sulfo-LC-SMPT 100 5624 GA 0.3% 614 GA 0.5% 322 SOLUBLE LIPASE 732

[0441] Table XXXI above demonstrates the effects of various crosslinkersand degrees of crosslinking on Candida rugosa lipase crystals. Theamino/sulfo reactive heterobifunctional crosslinker sulfo-LC-SMPTdramatically enhanced the activity of lipase (greater than 7 fold) to anolive oil substrate when compared to the soluble enzyme. The aminoreactive (DSP) or the sulfhydryl reactive (BMH) homobifunctionalcrosslinkers showed slightly enhancing and approximately 2-foldenhancement of activity, respectively, when compared to soluble enzyme.

Example 53 Reversible Crosslinkers—Disulfide Crosslinked Candida RugosaLipase Crystals

[0442]Candida rugosa lipase crystals were prepared as described inExample 48. Samples containing crystals of 20-30 μM in average size anda total protein of 42 mg/ml were crosslinked using either of thereversible crosslinkers:

[0443] (1) Dimethyl 3,3′-dithiobispropionimidate.HCl (DTBP) (Pierce), or

[0444] (2) Dithiobis (succinimidylpropionate)(DSP) (Pierce),

[0445] (3) Bis[2-(sulfosuccinimidooxycarbonyloxy)-ethyl]sulfone(sulfo-BSOCOES) (Pierce).

[0446] The crosslinking reaction was carried out in duplicate 1.5 mlmicrocentrifuge tubes (USA/Scientific) by placing 250 μl of lipasecrystal slurry (11.5 mg) into 500 μl of buffer containing 10 μM HEPES,10 mM calcium acetate, 20% MPD at pH 8.5. Next, the crosslinkingreaction was initiated by adding one crosslinker solution to each tubeas follows:

[0447] A) 50 mM DTBP—27.9 mg of DTBP was dissolved in 60 μl of water and20 μl of this solution was added to one tube with the crystals; and

[0448] B) 14.8 mM DSP—35.0 mg of DSP was dissolved in 120 μl of DMSO andthen 10 μl of this solution was added to one tube with the crystals.

[0449] C) 7.5 mM Sulfo-BSOCOES—7.2 mg of sulfo-BSOCOES was dissolved in60 μl of water and then 20 μl of this solution was added to one tubewith the crystals.

[0450] The tubes with DTBP and DSP were tumbled at ambient temperature(24-26° C.) for approximately 2 days or until the sample was determinedto be insoluble in 32 mM NaOH. The tube with sulfo-BSOCOES was tumbledat ambient temperature (24-26° C.) for approximately 2 days. Thesolubility test consisted of adding 50 μl of sample to 150 μl of 32 mMNaOH. In this test, uncrosslinked samples were readily soluble in 32 mMNaOH at the same concentrations. The crosslinking reaction wasterminated by centrifuging the sample at 3000 rpm for 5 minutes. Next,the supernatant was discarded and washed 3 times with 1 ml of 10 mMTris.HCl buffer containing 10 mM calcium chloride and 20% MPD at pH 7.0.

Example 54 Dissolution of Disulfide Bond—Containing Crosslinked CandidaRugosa Lipase Crystals

[0451] A 200 mM solution of cysteine was prepared by dissolving 242 mgof cysteine in 10 ml of 10 mM TRIS HCl buffer containing 10 mM calciumchloride and 20% MPD at pH 7. A 200 μl sample of crosslinked crystalslurry was centrifuged at 3000 rpm for 5 minutes and the supernatant wasdiscarded. The pellet was suspended in 200 μl of cysteine containingTRIS buffer. Another 200 μl of crosslinked sample was taken andcentrifuged at 3000 rpm for 5 minutes and the supernatant was discarded.This pellet was then suspended in 200 μl of 10 mM Tris.HCl buffer at pH7.0 without cysteine. All samples were incubated at 37° C. for 1 hourand monitored by direct visual and microscopic observation fordissolution in 32 mM NaOH.

[0452] The sample exposed to DTBP was fully soluble in the presence of200 mM cysteine and insoluble in its absence after incubation for 1 hourat 37° C. The DSP sample was slightly soluble after 1 hour in thepresence of cysteine and insoluble in its absence.

Example 55 Dissolution by Base Cleavable Crosslinked Candida RugosaLipase Crystals

[0453] A 200 μl sample of crosslinked crystal slurry was centrifuged at3000 rpm for 5 minutes and the supernatant was discarded. The pellet wassuspended in 200 μl of Tris buffer and 600 μl of 32 mM NaOH. Another 200μl of crosslinked sample was taken and centrifuged at 3000 rpm for 5minutes and the supernatant was discarded. This pellet was thensuspended in 200 μl of 10 mM Tris.HCl buffer at pH 7.0. All samples wereincubated at 37° C. for 1 hour and monitored by direct visual andmicroscopic observation for dissolution in 32 mM NaOH.

[0454] The sample exposed to sulfo-BSOCOES was fully soluble in thepresence of NaOH and insoluble in its absence.

[0455] While we have hereinbefore described a number of embodiments ofthis invention, it is apparent that our basic constructions can bealtered to provide other embodiments which utilize the processes andcompositions of this invention. Therefore, it will be appreciated thatthe scope of this invention is to be defined by the claims appendedhereto rather than by the specific embodiments which have been presentedhereinbefore by way of example.

We claim:
 1. A crosslinked protein crystal, said protein crystal beingcapable of change from insoluble and stable form to soluble and activeform and releasing between 0.1% and 100% of crystalline material assoluble protein per day upon a change in the environment surroundingsaid crystal, said change being selected from the group consisting of:change in temperature, change in pH, change in chemical composition,change from concentrate to dilute form, change in shear force actingupon the crystal and combinations thereof.
 2. The crosslinked proteincrystal according to claim 1, wherein said change from concentrate todilute form comprises a change in solute concentration.
 3. Thecrosslinked protein crystal according to claim 2, wherein said change insolute concentration comprises an increase or decrease in saltconcentration.
 4. The crosslinked protein crystal according to claim 3,wherein said change in solute concentration comprises a decrease in saltconcentration.
 5. The crosslinked protein crystal according to claim 2,wherein said change in solute concentration comprises an increase ordecrease in water concentration.
 6. The crosslinked protein crystalaccording to claim 5, wherein said change in solute concentrationcomprises an increase in water concentration.
 7. The crosslinked proteincrystal according to claim 2, wherein said change in soluteconcentration comprises an increase or decrease in organic solventconcentration.
 8. The crosslinked protein crystal according to claim 2,wherein said change in solute concentration comprises a decrease indetergent concentration.
 9. The crosslinked protein crystal according toclaim 2, wherein said change in solute concentration comprises adecrease in protein concentration.
 10. The crosslinked protein crystalaccording to claim 1, wherein said change from concentrate to diluteform comprises a change in concentration of all solutes from about2-fold to about 10,000-fold.
 11. The crosslinked protein crystalaccording to claim 10, wherein said change from concentrate to diluteform comprises a change in concentration of all solutes from about2-fold to about 700-fold.
 12. The crosslinked protein crystal accordingto claim 1, wherein said change in pH comprises a change from acidic pHto basic pH.
 13. The crosslinked protein crystal according to claim 1,wherein said change in pH comprises a change from basic pH to acidic pH.14. The crosslinked protein crystal according to claim 1, wherein saidchange in temperature comprises an increase or decrease in temperature.15. The crosslinked protein crystal according to claim 14, wherein saidchange in temperature is an increase in temperature from a lowtemperature between about 0° C. and about 20° C. to a high temperaturebetween about 25° C. and about 70° C.
 16. The crosslinked proteincrystal according to claim 1, wherein said active form of said proteinis a form which is active against macromolecular substrates.
 17. Acrosslinked protein crystal, said protein crystal having a half-life ofactivity under storage conditions which is greater than at least 2 timesthat of the soluble form of the protein that is crystallized to formsaid crystal that is crosslinked and activity similar to that of thesoluble form of the protein under conditions of use and which releasesbetween 0.1% and 100% of crystalline material as soluble protein perday.
 18. A crosslinked protein crystal, said protein crystal beingcapable of releasing its protein activity at a controlled rate ofbetween 0.1% and 100% of crystalline material as soluble protein per dayupon exposure to a change in the environment surrounding said crystal,said change being selected from the group consisting of change in pH,change in solute concentration, change in temperature, change inchemical composition, change in shear force acting upon the crystals andcombinations thereof.
 19. A crosslinked protein crystal, said proteincrystal having a 5 to 10 fold higher protein activity for any one of amacromolecular substrate, a biphasic substrate or a small moleculesubstrate, as compared with the soluble form of the protein that iscrystallized to form the crystals that are crosslinked and releasingbetween 0.1% and 100% of crystalline material as soluble protein perday.
 20. A crosslinked protein crystal, said protein crystal having a 2to 3 fold higher protein activity for any one of a macromolecularsubstrate, a biphasic substrate or a small molecule substrate ascompared with the soluble form of the protein that is crystallized toform the crystals that are crosslinked and releasing between 0.1% and100%c of crystalline material as soluble protein per day.
 21. Acrosslinked protein crystal, said protein crystal having at least 2times higher protein activity for a macromolecular substrate, a biphasicsubstrate or a small molecule substrate as compared with the solubleform of the enzyme that is crystallized to form the crystals that arecrosslinked and releasing between 0.1% and 100% of crystalline materialas soluble protein per day.
 22. A crosslinked lipase crystal, saidlipase crystal having a 5 to 10 fold higher enzyme activity forhydrolysis of a biphasic olive oil substrate as compared with thesoluble form of the lipase that is crystallized to form the crystalsthat are crosslinked.
 23. The crosslinked lipase crystal according toclaim 22, said lipase crystal having a 2 to 3 fold higher enzymeactivity for hydrolysis of a biphasic olive oil substrate as comparedwith the soluble form of the lipase that is crystallized to form thecrystals that are crosslinked.
 24. The crosslinked lipase crystalaccording to claim 23, said lipase crystal having at least 2 timeshigher protein activity for hydrolysis of a biphasic olive oil substrateas compared with the soluble form of the lipase that is crystallized toform the crystals that are crosslinked.
 25. The crosslinked proteincrystal according to any one of claims 22, 23 or 24, wherein saidprotein is crosslinked bysulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido] hexanoate(Sulfo-LC-SMPT).
 26. The crosslinked protein crystal according to anyone of claims 22, 23 or 24, wherein said protein is crosslinked by1-ethyl-3-[3-dimethylaminoproplyl]carbodiimide hydrochloride (EDC). 27.The crosslinked lipase crystal according to any one of claims 22, 23 or24, wherein said lipase is crosslinked bysulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido] hexanoate(Sulfo-LC-SMPT).
 28. The crosslinked lipase crystal according to any oneof claims 22, 23 or 24, wherein said lipase is crosslinked by1-ethyl-3-[3-dimethylaminoproplyl] carbodiimide hydrochloride (EDC). 29.The crosslinked protein crystal according to any one of claims 1, 17,18, 19, 20 or 21, wherein said protein is crosslinked by ahomobifunctional crosslinker.
 30. The crosslinked protein crystalaccording to any one of claims 1, 17, 18, 19, 20, or 21, wherein saidprotein is crosslinked by a heterobifunctional crosslinker.
 31. Thecrosslinked protein crystal according to claim 18, wherein saidcontrolled rate of releasing protein activity is determined by a factorselected from the group consisting of: the degree of crosslinking ofsaid crosslinked protein crystal, the length of time of exposure ofprotein crystal to the crosslinker, the amino acids residues involved inthe crosslinks, whether the crosslinker is homobifunctional orheterobifunctional, the rate of addition of the crosslinking agent tosaid protein crystal, the nature of the crosslinker, the chain length ofthe crosslinker, the surface area of said crosslinked protein crystal,the size of said crosslinked protein crystal, the shape of saidcrosslinked protein crystal and combinations thereof.
 32. Thecrosslinked protein crystal according to claim 18, wherein said crystalhas a protein activity release rate of between about 0.1% per day andabout 100% per day.
 33. The crosslinked protein crystal according toclaim 18, wherein said crystal has a protein activity release ratebetween about 0.01% per hour and about 100% per hour.
 34. Thecrosslinked protein crystal according to claim 18, wherein said crystalhas a protein activity release rate between about 1% per minute andabout 50% per minute.
 35. The crosslinked protein crystal according toany one of claims 1, 17, 18, 19, 20 or 21, said protein crystal beingsubstantially insoluble and stable in a composition under storageconditions and substantially soluble and active under conditions of useof said composition.
 36. The crosslinked protein crystal according toclaim 35, wherein said composition is selected from the group consistingof cleaning agents, detergents, personal care compositions, cosmetics,pharmaceuticals, veterinary compounds, vaccines, foods, feeds,diagnostics and formulations for decontamination.
 37. The crosslinkedprotein crystal according to claim 36, wherein said detergent isselected from the group consisting of powdered detergents, liquiddetergents, bleaches, household cleaners, hard surface cleaners,industrial cleaners, carpet shampoos and upholstery shampoos.
 38. Thecrosslinked protein crystal according to claim 36, wherein said cosmeticis selected from the group consisting of creams, emulsions, lotions,foams, washes, gels, compacts, mousses, sunscreens, slurries, powders,sprays, foams, pastes, ointments, salves, balms, shampoos, and drops.39. The crosslinked protein crystal according to any one of claims 1,17, 18, 19, 20 or 21, wherein said protein is an enzyme.
 40. Thecrosslinked protein crystal according to claim 39, wherein said enzymeis selected from the group consisting of hydrolases, isomerases, lyases,ligases, transferases and oxidoreductases.
 41. The crosslinked proteincrystal according to claim 40, wherein said enzyme is selected from thegroup consisting of proteases, amylases, cellulases, lipases andoxidases.
 42. The crosslinked protein crystal according to any one ofclaims 1, 17, 18, 19, 20 or 21, wherein said protein is selected fromthe group consisting of therapeutic proteins, cleaning agent proteins,personal care proteins, veterinary proteins, food proteins, feedproteins, diagnostic proteins and decontamination proteins.
 43. Thecrosslinked protein crystal according to any one of claims 1, 17, 18,19, 20 or 21, wherein said protein is selected from the group consistingof hormones, antibodies, inhibitors, growth factors, trophic factors,cytokines, lymphokines, growth hormones, nerve growth hormones, bonemorphogenic proteins and toxoids.
 44. The crosslinked protein crystalaccording to any one of claims 1, 17, 18, 19, 20 or 21, wherein saidprotein is selected from the group consisting of insulin, amylin,erythropoietin, Factor VIII, TPA, dornase-α, α-1-antitripsin, urease,fertility hormones, FSH, LSH, postridical hormones, tetanus toxoid anddiptheria toxoid.
 45. The crosslinked protein crystal according to anyone of claims 1, 17, 18, 19, 20 or 21, said crystal having a longestdimension of between about 0.01 μm and about 500 μm.
 46. The crosslinkedprotein crystal according to any one of claims 1, 17, 18, 19, 20 or 21,said crystal having a longest dimension of between about 0.1 μm andabout 50 μm.
 47. The crosslinked protein crystal according to any one ofclaims 1, 17, 18, 19, 20 or 21, said crystal having a shape selectedfrom the group consisting of: spheres, needles, rods, plates, rhomboids,cubes, bipryamids and prisms.
 48. A composition comprising a crosslinkedprotein crystal according to any one of claims 1, 17, 18, 19, 20 or 21,said composition being selected from the group consisting of cleaningagents, detergents, personal care compositions, cosmetics,pharmaceuticals, veterinary compounds, vaccines, foods, feeds,diagnostics and formulations for decontamination.
 49. The compositionaccording to claim 48, wherein said detergent is selected from the groupconsisting of powdered detergents, liquid detergents, bleaches,household cleaners, hard surface cleaners, industrial cleaners, carpetshampoos and upholstery shampoos.
 50. The composition according to claim48, wherein said cosmetic is selected from the group consisting ofcreams, emulsions, lotions, foams, washes, gels, compacts, slurries,powders, sprays, foams, pastes, ointments, salves, balms, shampoos,sunscreens and drops.
 51. A protein delivery system, said systemcomprising crosslinked protein crystals according to any one of claims1, 17, 18, 19, 20, or
 21. 52. The protein delivery system according toclaim 51, wherein said protein is selected from the group consisting of:detergent enzymes, cosmetic proteins, pharmaceutical proteins,agricultural proteins, vaccine proteins and decontamination proteins.53. The protein delivery system according to claim 52, said proteindelivery system being a microparticulate protein delivery system. 54.The protein delivery system according to claim 53, wherein saidmicroparticulate protein delivery system comprises crosslinked proteincrystals having a longest dimension between about 0.01 μm and about 500μm.
 55. The protein delivery system according to claim 54, wherein saidmicroparticulate protein delivery system comprises crosslinked proteincrystals having a longest dimension of between about 0.1 μm and about 50μm.
 56. The protein delivery system according to claim 53, wherein saidmicroparticulate protein delivery system comprises crosslinked proteincrystals having a shape selected from the group consisting of: spheres,needles, rods, plates, rhomboids, cubes, bipryamids and prisms.
 57. Adetergent formulation comprising a crosslinked protein crystal accordingto any one of claims 1, 17, 18, 19, 20, or
 21. 58. A controlled releaseformulation comprising a crosslinked protein crystal according to anyone of claims 1, 17, 18, 19, 20 or
 21. 59. A pharmaceutical controlledrelease formulation comprising a crosslinked protein crystal accordingto any one of claims 1, 17, 18, 19, 20 or
 21. 60. A pharmaceuticalcontrolled release formulation comprising a crosslinked protein crystal,said crystal being substantially insoluble under storage conditions andcapable of releasing its protein activity in vivo at a controlled rateof between 0.1% and 100% of crystalline material as soluble protein perday.
 61. The pharmaceutical controlled release formulation according toclaim 59, said pharmaceutical being capable of administration byparenteral or non-parenteral routes.
 62. The pharmaceutical controlledrelease formulation according to claim 61, said pharmaceutical beingcapable of administration by oral, pulmonary, nasal, aural, anal,dermal, ocular, intravenous, intramuscular, intraarterial,intraperitoneal, mucosal, sublingual, subcutaneous or intracranialroute.
 63. The pharmaceutical controlled release formulation accordingto claim 59, wherein said pharmaceutical is capable of administration byoral route and said crosslinked protein crystal is substantiallyinsoluble under gastric pH conditions and substantially soluble undersmall intestine pH conditions.
 64. A vaccine comprising a crosslinkedprotein crystal according to any one of claims 1, 17, 18, 19, 20 or 21.65. A formulation comprising a crosslinked protein crystal according toany one of claims 1, 17, 18, 19, 20 or 21, said formulation beingselected from the group consisting of tablets, liposomes, granules,spheres, microspheres, microparticles and capsules.
 66. A method forproducing crosslinked protein crystals comprising the step of reactingprotein crystals with a first crosslinking agent, or a firstcrosslinking agent and at least a second crosslinking agent, underconditions sufficient to induce crosslinking of said crystals to theextent that the resulting crosslinked crystals are characterized by theability to change from insoluble and stable form to soluble and activeform upon a change in their environment, and to release between 0.1% and100% of crystalline material as soluble protein per day, wherein saidchange is selected from the group consisting of change in temperature,change in pH, change in chemical composition, change from concentrate todilute form, change in shear force acting upon the crystals andcombinations thereof.
 67. A method for producing crosslinked proteincrystals comprising the step of reacting protein crystals with a firstcrosslinking agent, or a first crosslinking agent and at least a secondcrosslinking agent, under conditions sufficient to induce crosslinkingof said crystals to the extent that the resulting crosslinked crystalsare characterized by a half-life of activity under storage conditionswhich is greater than at least 2 times that of the soluble form of theprotein that is crystallized to form said crystals that are crosslinkedand activity similar to that of the soluble form of the protein andwhich release between 0.1% and 100% of crystalline material as solubleprotein per day under conditions of use.
 68. A method for producingcrosslinked protein crystals comprising the step of reacting proteincrystals with a first crosslinking agent, or a first crosslinking agentand at least a second crosslinking agent, under conditions sufficient toinduce crosslinking of said crystals to the extent that the resultingcrosslinked crystals are characterized by being capable of releasingtheir protein activity at a controlled rate of between 0.1% and 100% ofcrystalline material as soluble protein per day upon exposure to achange in their environment, said change being selected from the groupconsisting of change in pH, change in soluble concentration, change intemperature, change in chemical composition, change in shear forceacting upon the crystals and combinations thereof.
 69. The method forproducing crosslinked protein crystals according to any one of claims66, 67 or 68, comprising the step of reacting said protein crystals withsaid first crosslinking agent and said at least a second crosslinkingagent at the same time or in sequence.
 70. The method for producingcrosslinked protein crystals according to any one of claims 66, 67 or68, wherein, prior to reacting protein crystals with said crosslinkingagent, said method further comprises the step of crystallizing saidprotein.
 71. The method for producing crosslinked protein crystalsaccording to any one of claims 66, 67 or 68, wherein the conditionssufficient to induce crosslinking are dependent upon a factor selectedfrom the group consisting of: the degree of crosslinking of saidcrosslinked protein crystals, the length of time of exposure of proteincrystals to the crosslinking agent, the rate of addition of thecrosslinking agent to said protein crystal, the nature of thecrosslinker, the chain length of the crosslinker, the surface area ofsaid crosslinked protein crystals, the size of said crosslinked proteincrystals, the shape of said crosslinked protein crystals andcombinations thereof.
 72. The method for producing crosslinked proteincrystals according to claim 68, wherein said controlled rate ofreleasing protein activity is determined by a factor selected from thegroup consisting of: the degree of crosslinking of said crosslinkedprotein crystals, the length of time of exposure of protein crystals tothe crosslinking agent, the rate of addition of the crosslinking agentto said protein crystals, the nature of the crosslinker, the chainlength of the crosslinker, the surface area of said crosslinked proteincrystals, the size of said crosslinked protein crystals, the shape ofsaid crosslinked protein crystals and combinations thereof.
 73. Themethod for producing crosslinked protein crystals according to any oneof claims 66, 67 or 68, wherein said crosslinking agent is amultifunctional crosslinking agent.
 74. The method for producingcrosslinked protein crystals according to claim 73, wherein saidcrosslinking agent is a bifunctional crosslinking agent.
 75. The methodfor producing crosslinked protein crystals according to claim 73,wherein said crosslinking agent is selected from the group consistingof: glutaraldehyde, succinaldehyde, octanedialdehyde and glyoxal. 76.The method for producing crosslinked protein crystals according to claim73, wherein said crosslinking agent is selected from the groupconsisting of: halo-triazines, halo-pyrimidines, anhydrides of aliphaticor aromatic mono- or di-carboxylic acids, halides of aliphatic oraromatic mono- or di-carboxylic acids, N-methylol compounds,di-isocyanates, di-isothiocyanates and aziridines.
 77. The method forproducing crosslinked protein crystals according to any one of claims66, 67 or 68, wherein said crosslinking agent is an epoxide.
 78. Themethod for producing crosslinked protein crystals according to claim 77,wherein said epoxide is selected from the group consisting of: neopentylglycol diglycidyl ether, ethylene glycol diglycidyl ether, di-epoxides,tri-epoxides and tetra-epoxides.
 79. The method for producingcrosslinked protein crystals according to any one of claims 66, 67 or68, wherein said crosslinking agent is 0.0076% to 0.5% glutaraldehydeand wherein the conditions sufficient to induce crosslinking includereacting protein crystals with a crosslinking agent for a period of timebetween about 3 minutes and about 120 minutes.
 80. The method forproducing crosslinked protein crystals according to claim 79, whereinsaid crosslinking agent is 0.005% glutaraldehyde and wherein theconditions sufficient to induce crosslinking include reacting proteincrystals with a crosslinking agent for a period of time between about 10minutes and about 30 minutes.
 81. The method for producing crosslinkedprotein crystals according to claim 79 wherein, prior to reaction withsaid protein crystals, said glutaraldehyde is pretreated by incubationat 60° C. with a buffer for 1 hour.
 82. The method for producingcrosslinked protein crystals according to any one of claims 66, 67 or68, wherein said crosslinking agent is 0.01% to 1% glyoxal and whereinthe conditions sufficient to induce crosslinking include reactingprotein crystals with a crosslinking agent for a period of time betweenabout 30 minutes and about 60 minutes.
 83. The method for producingcrosslinked protein crystals according to any one of claims 66, 67 or68, wherein said crosslinking agent is 0.05% to 1% octanedialdehyde andwherein the conditions sufficient to induce crosslinking includereacting protein crystals with a crosslinking agent for a period of timebetween about 30 minutes and about 16 hours.
 84. The method forproducing crosslinked protein crystals according to claim 83, whereinsaid crosslinking agent is 1% octanedialdehyde and wherein theconditions sufficient to induce crosslinking include reacting proteincrystals with a crosslinking agent for a period of time between about 1hour and about 3 hours.
 85. The method for producing crosslinked proteincrystals according to any one of claims 66, 67 or 68, wherein saidcrosslinking agent is 1% succinaldehyde and wherein the conditionssufficient to induce crosslinking include reacting protein crystals witha crosslinking agent for a period of time between about 30 minutes andabout 3 hours.
 86. The method for producing crosslinked protein crystalsaccording to any one of claims 66, 67 or 68, wherein said firstcrosslinking agent is 0.01% to 4% epoxide and said second crosslinkingagent is 0.1% to 0.2% glutaraldehyde and wherein the conditionssufficient to induce crosslinking include reacting said protein crystalswith said first crosslinking agent for a period of time between about 1hour and about 72 hours and reacting said protein crystals with saidsecond crosslinking agent for a period of time between about 1 hour andabout 5 hours.
 87. The method for producing crosslinked protein crystalsaccording to claim 86, wherein said first crosslinking agent is 0.01%epoxide and said second crosslinking agent is 0.1% glutaraldehyde andwherein the conditions sufficient to induce crosslinking includereacting said protein crystals with said first crosslinking agent forabout 5 hours and reacting said protein crystals with said secondcrosslinking agent for about 1.5 hours.
 88. The method for producingcrosslinked protein crystals according to any one of claims 66, 67 or68, wherein said protein is an enzyme.
 89. The method for producingcrosslinked protein crystals according to any one of claims 66, 67 or68, wherein said crosslinking agent is a reversible crosslinking agent.90. The method for producing crosslinked protein crystals according toclaim 89, wherein said reversible crosslinking agent is a disulfidecrosslinking agent.
 91. The method for producing crosslinked proteincrystals according to claim 90, wherein said disulfide crosslinkingagent is a homobifunctional crosslinking agent or a heterobifunctionalcrosslinking agent.
 92. The method for producing crosslinked proteincrystals according to any one of claims 66, 67 or 68, wherein saidprotein is an enzyme.
 93. The method for producing crosslinked proteincrystals according to claim 92, wherein said enzyme is selected from thegroup consisting of hydrolases, isomerases, lyases, ligases,transferases and oxidoreductases.
 94. The method for producingcrosslinked protein crystals according to claim 93, wherein said enzymeis from the group consisting of proteases, amylases, cellulases, lipasesand oxidases.
 95. The method for producing crosslinked protein crystalsaccording to any one of claims 66, 67 or 68, wherein said protein isselected from the group consisting of therapeutic proteins, cleaningagent proteins, personal care proteins, veterinary proteins, foodproteins, feed proteins, diagnostic proteins and decontaminationproteins.
 96. The method for producing crosslinked protein crystalsaccording to claim 95, wherein said protein is selected from the groupconsisting of hormones, antibodies, inhibitors, growth factors, trophicfactors, cytokines, lymphokines, growth hormones, nerve growth hormones,bone morphogenic proteins and toxoids.
 97. The method for producingcrosslinked protein crystals according to claim 96, wherein said proteinis selected from the group consisting of insulin, amylin,erythropoietin, Factor VIII, TPA, dornase-α, α-1-antitripsin, urease,fertility hormones, FSH, LSH, postridical hormones, tetanus toxoid anddiptheria toxoid.